JP2003047461A - Method for carrying out high-density cell culture of cell by using apatite sheet, culturing device and cell culture module - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for carrying out high-density cell culture of a cell by using an apatite sheet and to provide a culturing device used therefor. SOLUTION: A cell culture module utilizing an apatite sheet for cell culture bed was newly developed in the present invention. In the module, a culture medium is fed so as to pass through the apatite sheet for cell culture bed built in a culture device and cells are proliferated also in the interior of the sheet. This utilization method uses continuous spaces even between fibers of the apatite sheet.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apatite sheet.
The present invention relates to a high-density cell culture method, a culture device, and a culture module for cells using cells, particularly animal cells. More specifically, the present invention provides a high-density cell culture method for culturing animal cells at high density, particularly animal cells that produce physiologically active substances,
The present invention relates to a culture device and a culture module.

[0002]

[Prior Art] In the latter half of the 20th century, important discoveries were made in the life sciences, led by the elucidation of the molecular structure of DNA. Based on these discoveries, biotechnology such as gene manipulation rapidly developed. With the progress of biotechnology,
Various materials have been developed to support it.
Typical examples thereof include polystyrene plastic culture dishes and microplates used for culturing microorganisms or cells. Microplates are widely used as an indispensable instrument for carrying out enzyme-linked immunosorbent assay (ELISA), which is indispensable for ultra-quantitative analysis of biological components. Polymer beads used as polymer gels for electrophoresis and column packing materials for high performance liquid chromatography played an extremely large role in the separation and analysis of biological components such as nucleic acids and proteins. Many of the ingredients of these materials are bio-derived polysaccharides such as agarose and dextran. In addition, there are nitrocellulose film and PVDF film used for hybridization, which is a gene analysis technology, and the use of these films has increased the gene analysis speed.
In addition to organic polymer materials, inorganic materials are also widely used. There are glass beads as an adsorbent material for DNA recovery and glass fiber as a cell attachment material. It can be said that the development of new materials has promoted the development of biotechnology.

Hydroxyapatite Hydroxyapatite as a material related to biotechnology
(Hereinafter abbreviated as apatite or HAp). HAp
Belongs to the hexagonal crystal system with a chemical composition of A ₁₀ (MO ₄ ) ₆ X ₂ and is a calcium phosphate-based inorganic material with a composition of Ca ₁₀ (PO ₄ ) ₆ (OH) _2. is there. In a broad sense, the composition of A ₁₀ (MO ₄ ) ₆ X ₂ is also called apatite (Ap), but in this paper, apatite is hydroxyapatite (HAp). Calcium phosphate has fluoroapatite Ca ₁₀ (PO ₄ ) ₆ F _2, which is the main constituent of phosphate rock and the only industrial resource of phosphorus. On the other hand, HAp is known as a biomineral structure material for animal bones and teeth. A synthetic method for apatite has been developed, and the applications of synthetic apatite are expanding to biocompatible materials, ion exchangers, sensors and so on.

[0004] Research into utilizing HAp as a medical artificial bone material by utilizing its biocompatibility property is ongoing [Monr
oe EA, V otava W., Bass DB and McMullen J., J.
Dent. Res., 50 (4), 860 (1971); Aoki H., Kato
K., Ceramics, 10, 469 (1975); Denissen HW, Groo
t K., Makkers et al., J. Biomed. Mater. Res., 14,7
13 (1980); Groot K., J. Biomaterials, 1, 47 (198
0); Akao M., Ceramics, 20, 1 096 (1985); Suzuki
T., Gipusum & Lime, 195, 87 (1989); Di Silvio L.,
Dalby M. and Bonfield W., J. Materials Science Ma
terials in Medicine, 9, 845 (1998); Labat B., Dem
onet N., Rattner A., Aurelle JL, Rieu J., Frey
J. and Chamson A., J. Biomed. Mater. Res., 4 6, 33
1 (1999)].

[0005] In addition, various components (heavy metal ions, fluoride ions, biological levels) related to the living body due to the anion site (PO 4 ^3- site, OH ^- site) and cation site (Ca ²⁺ site) of HAp The use of molecules as adsorbents or ion exchangers has also been studied [Bernardi
G., Methods of Enzymology, XXI, Academic Press,
(WB Jakoby, Ed.) New York, 95 (1971)]. As a method of using it as an adsorbent for biopolymers (proteins, nucleic acids, sugars), it has been studied to use it as a packing material for high performance liquid chromatography [Bernardi G., Methods of Enzymolo
gy, XXI, AcademicPress, (WB Jakoby, Ed.) New Yor
k, 95 (1971); Bernardi G., Giro M. and Gaillard
C., Biochim. Biophys. Acta, 278, 409 (1972); Spenc
er M. and Grynpas M., J. Chromatography, 1 66, 423
(1978); Moss B. and RosenblumN., J. Biol. Che
m., 247, 5194 (1972); Atkinson A., Bradford PA
and Selmes IP, J. Appl. Chem. Biotechnol., 23, 5
17 (1973); Kawasaki T., J. Chromatogr., 151, 95 (1
978)]. Focusing on the aspect of the morphology of those materials, HAp powder was processed into a shape similar to teeth and bones, and a bead shape was formed for the purpose of filling into a ceramic molded body or column made by sintering. limited.

Cell-adhesive materials have been studied by taking advantage of the biocompatibility of HAp, but the morphology is ceramicized beads or plates, and has not spread (Suzuki T., Toriyama). M., Kawamoto Y., Yo
kogawa Y. and Kawamura S., J. Ferment. Bioeng. ， 7
0, 164, (1990): Satoru Mizutani, "The world of bioreactors"
(Takeshi Kobayashi and others), Hario Research Institute, 259, (1992)).

On the other hand, there are many kinds of sheet-shaped and film-shaped materials as biotechnology-related materials, but there is no material in which HAp is molded into a sheet shape. If HAp can be formed into a sheet material, it must be used extensively in other biotechnology fields by taking advantage of its properties. For example, protein / nucleic acid adsorption sheets, virus adsorption mask substrate sheets, cell culture sheets, etc. can be expected. Furthermore, in the biotechnology industry, there is a high possibility that new biotechnology using sheet-shaped HAp materials will be developed. This is
It also means that apatite sheets will become a new material to support biotechnology.

Further, the development of an apatite sheet is the creation of a new functional paper in the field of paper processing products. HA
You can use p to add new added value to paper. If HAp can be processed into sheet material, H
The new use of Ap will be expanded and the economic effect will be great. This sheet has the potential to be used not only in the bio-related industry but also for household use. For example, it would be possible to use the HAp sheet as wallpaper that has the function of cleaning the interior space by taking advantage of the ion adsorption characteristics. If this happens, it will be necessary to develop manufacturing technology for apatite sheets in order to create new paper products that use HAp and open up new demand. However, there have been examples of development of needle-shaped crystals of HAp or sintered fibers [Iwas
aki H. and Kaneko Y., Zairyo, 37, 60 (1988); Ki
noshita M., Kishioka A., Hayashi H., Itatani K., G
ipusum & Lime, 219, 23 (1989)], a practical apatite monofilament that can be used as a raw material for sheets has not been produced. Further, an apatite sheet obtained by making apatite single fibers into a sheet by using a papermaking technique has not been developed either.

[0009] Therefore, the inventors of the present invention aim to develop a new sheet material using apatite as a raw material in order to promote the further utilization of HAp to promote the bio-related industry and to promote a Japanese paper company. It was As a result, monofilament-shaped HA
We succeeded in creating a new apatite sheet manufacturing technology using the papermaking method, which is a paper manufacturing technology, by newly developing p. That is, the inventors of the present invention have devised a new method of producing an apatite sheet using a papermaking method that is a method of making paper using the HAPC prepared above as a raw material fiber. This method is completely different from the conventional method of fixing the inorganic powder to the pulp using a polymeric fixing agent and making it into a sheet, which is low cost and labor saving without using a fixing agent. It is the method that was done.

That is, a method for synthesizing a composite fiber in which HAp and pulp are integrated has been described (Kawakatsu Hironobu, Functional Paper Study Group, 32, 28 (1993)). That is, HAp is synthesized on the pulp surface using the wet synthesis reaction and insoluble H
Apatite / pulp composite fiber (hereinafter HA
We have developed a method of creating a computer (abbreviated as PC). This HAPC can be called HAp monofilament if its surface characteristics are focused. Up until now, HAp has been developed as a ceramic fiber, but in reality it was a fiber material with little practicality. In other words, by combining HAp with pulp, it was possible to obtain a highly practical fiber material as an organic / inorganic composite fiber. It was also confirmed that the apatite sheet exhibits excellent performance as a protein / nucleic acid adsorption material.

[0011]

The present inventors have also found that
A novel apatite sheet for animal cell culture beds was developed using HAPC as the raw material fiber. As a result, we found that HA
We have developed an apatite sheet for cell culture beds, which is a sheet of PC and has excellent cell adhesiveness and can grow cells at high density. By examining mainly the surface characteristics or the internal structure of the sheet as a culture bed, we devised a method of statically culturing CHO cells using a sheet and a method of rotating culture in a spinner flask. It was found that animal cells such as CHO cells can be easily and efficiently cultured by this method.

The high-density cell culturing method according to the present invention enables high-density cell culturing for a long period of time, and it is useful to utilize cytokines including interleukins and the like by utilizing a culturing apparatus having an apatite sheet incorporated in a module. It has become possible to efficiently culture cells containing physiologically active substances, and as a result, it has become possible to efficiently produce such useful physiologically active substances.

[0013]

DISCLOSURE OF THE INVENTION The present invention provides an apatite sheet for an animal cell culture bed as a material for supporting biotechnology, and provides a high-density cell culture method for animal cells using the apatite sheet. With the goal.

Another object of the present invention is to provide a novel module capable of high-density cell culture, which incorporates an apatite sheet for animal cell culture beds.

Further, another object of the present invention is to provide a highly efficient cell culture system using a module capable of high-density cell culture incorporating the apatite sheet.

Furthermore, the present invention provides a useful physiologically active substance comprising efficiently producing a useful physiologically active substance by efficiently culturing animal cells at a high density by a high density cell culture method for animal cells. Another purpose is to provide a production method.

[0017]

In order to achieve this object, the present invention uses a culture device in which an apatite sheet is arranged and incorporated so that its surface area is large, and cells containing animal cells are A high-density cell culture method for cells using an apatite sheet, which comprises culturing at high density.

In a preferred embodiment of the present invention, cells can be cultured at high density by continuously culturing the cells. In a further preferred embodiment, animal cells can be cultivated at high density by continuously culturing animal cells. In a further preferred embodiment, the present invention enables efficient cultivation of useful physiologically active substances by continuously culturing animal cells as cells.

The present invention also provides a culture device incorporating a novel animal cell culture module utilizing the apatite sheet for cell culture beds.

Furthermore, the present invention provides a cell culture module using a cell culture bed apatite sheet.

The present invention provides an animal cell culture module that can be incorporated into a cell continuous culture device capable of continuous culture of animal cells such as CHO-K1 cells.

[0022]

BEST MODE FOR CARRYING OUT THE INVENTION First, an apatite-pulp composite will be described.

HAp can be synthesized by various methods. The methods include a dry synthesis method and a wet synthesis method (Boskey
AL and Posner AS, J. Phs. Chem., 80, 40 (197
6); Moreno EC, Gregory TM and Brown WE, J. R.
es. Natl. Bur. Stand., 72A, 773 (1968); McDowell
M., Gregory TM and Brown WE, J. Res. Natl. Bu
r.Stand., 81A, 273 (1977)), hydrothermal method (Hayek
W., Bohler W., Leuchlutter J. and Petter H., Z. An
org. Allg. Chem., 295, 24 (1958); Kirn JF and L
eidheiser H., J. Crystal Growth, 2, 111 (1968); Ey
sel W. and Roy DM, J. Crystal Growth, 20, 245 (1
973)).

In the dry synthesis method, HAp is obtained by mixing appropriate calcium salt and phosphate in a Ca / P ratio of 5: 3 and heating at 1000 ° C. or higher in a steam atmosphere. For example, HAp can be obtained by mixing Ca ₃ (PO ₄ ) ₂ and CaO and heating in a steam atmosphere.

The wet synthesis method is a method of precipitating HAp by mixing aqueous solutions of Ca ²⁺ and PO 4 ³⁻ . For example, HAp can be obtained by precipitation from an aqueous solution of ammonium dihydrogen phosphate and an aqueous solution of calcium nitrate, followed by aging, washing by boiling, etc. and drying. HAp synthesized by the wet method is extremely fine and has good dispersibility, but it is difficult to dehydrate because it has a gel form. Furthermore, there is a drawback that the characteristics of the product change due to many factors such as the type of raw material, concentration, mixing procedure, control of speed pH.

The present inventor utilizes the HAp wet synthesis reaction to synthesize a HAp insoluble crystalline salt synthesis reaction on the pulp surface to synthesize an apatite-pulp composite (HAPC) in which the pulp surface is coated with HAp. (Kawakatsu Hironobu, Functional Paper Research Society, 32, 28 (1993)). At that time, it was clarified that the amount of HAp deposited can be arbitrarily changed by changing the concentration of the raw material in the synthesis conditions.
HAPC is a functional pulp that is a composite of pulp and HAp, which is an inorganic powder, and is a pulp fiber with various HAp characteristics. From another point of view, HAPC made a single fiber of apatite. Since this HAPC has a pulp shape, it can be used for various purposes.

On the other hand, HAp ceramic monofilaments have been used so far in Iwasaki et al. (Iwasaki H. and KanekoY., Zairyo, 37, 60).
(1988)) or Kinoshita et al. (Kinoshita M., Kishioka)
A., Hayashi H., Itatani K., Gipusum & Lime, 21
9, 23 (1989)). Iwasaki et al. Used a mixed solution of calcium nitrate, diammonium hydrogen phosphate and urea as a raw material,
Reflux in a silicone oil bath at temperatures of 95 ° C, 95 ° C and 100 ° C. As a result, whiskers or fibrous carbonate apatite were synthesized, and ceramic HAp fibers were obtained by sintering the fibers at 1200 ℃. The size of the fibers was several μm in diameter and the length was 20 to 150 μm. On the other hand, Kinoshita et al. Used a solution of sodium pyrophosphate dissolved in a sodium alginate solution as a starting material. This starting material was spun into a spinning solution (mixed solution of calcium chloride and calcium acetate) from a spinning machine to prepare gel fibers. The fibers are aged, washed with water, dried, and fired at 900 ° C for 1 hour in air to obtain fibrous ceramic HAp. Fiber size is diameter 5
It can be said that 0 μm and the length can be adjusted to some extent.
However, since both fibers are ceramic fibers, they have the drawbacks of poor flexibility and brittleness. Sheet materials have been trial-produced using these fibers as raw materials, but they are considered to have poor practicality. Compared to that,
HAPC has a certain degree of suppleness because the pulp is the core, and it is a material that is easy to handle, and is practical enough to be formed into a sheet.

The method for producing an apatite sheet by a papermaking method using HAPC as a raw material fiber is to make a sheet of HAp simply and at low cost, as compared with the production method of the inorganic powder blended paper which has been performed so far. You can Furthermore, since this manufacturing method applies a papermaking method, it is easy for small and medium-sized Japanese paper companies to introduce this technology. It was also clarified that the apatite sheet has excellent performance as a material for adsorbing proteins and nucleic acids.

Apatite has been studied as a carrier for culturing animal cells because of its biocompatibility. Among them, the use as ceramic beads and ceramic plates has been reported (Suzuki T., Toriyama M., Kawam.
oto Y., Yokogawa Y. and Kawamura S., J. Ferment. B
ioeng. , 70, 164, (1990); Satoru Mizutani, "The world of bioreactors" (Takeshi Kobayashi et al.), Hario Research Institute, 259, (199)
2)). However, there is no example of research on a culture bed in which apatite fiber is formed into a sheet. It is presumed that this is because it has been difficult to manufacture apatite fibers and it has not been possible to manufacture apatite sheets using them as a raw material. On the other hand, research is being conducted on the use of fibrous materials such as polyester fiber nonwoven fabric as a culture bed. Among them, fibrous materials have advantages over bead-shaped carriers, such as large adhesion surface area, easy handling, and easy molding (Perry SD and Wang D.
IC, Biotechnol. Bioeng., 34, NO.1, 1 (1989))),
A culture device that uses polyester fiber nonwoven fabric as a culture bed has also been devised (Mituda S., Matsuda Y., It
agaki Y., Suzuki A., Kumazawa E., Higashio K. and
Kawanishi G., J. Ferment. Bioeng. ， 70，289 (199
0); Robert J., Cote J., and Archamsbault, J. Biote
chnol. Bioeng. , 39, 697, (1992)). However,
Nonwoven fabric materials still have problems such as low affinity of raw material fibers for cells, and the development of sheet-shaped culture beds with excellent cell attachment and growth properties is still in progress.

It is considered that the obtained apatite sheet is a sheet-like material and, at the same time, has a high affinity for animal cells due to the characteristics of the apatite on the surface. In this respect,
It was found that, unlike the non-woven fabric made of chemical fiber, which has been considered as a culture bed up to now, it exhibits a superior cell growth performance.

Cell culture bed apatite sheet and sheet NP
The pore volumes of -0 are 2.86 and 1.71 cc / g, respectively,
The volume of the former is 1.7 times larger than that of the latter. The distribution of pore diameters is said to be suitable for animal cells to attach and proliferate. Areas with pore diameters of 10 to 200 μm (Kimiaki Yasuda, “The world of bioreactors” (Takeshi Kobayashi et al.), Hario) Institute,
62 (1992)), and the cell culture bed apatite sheet has excellent pore characteristics as a cell attachment material. As the pore volume increases, the surface area also increases and the sheet
The value was 0.32 m ² / g, 1.8 times that of NP-0. The difference in the pore volume and surface area of the two types of apatite sheets (sheet NP-0 and sheet NP-1) depends on the degree of beating of the raw pulp, and HAPC made from the raw beating pulp is used as the raw material. The smaller the pore volume, the smaller the volume. It can be seen that the pore volume and surface area of an apatite sheet are determined by the degree of beating of pulp, similar to pulp paper. The cell culture bed apatite sheet using the same HAPC as the raw material has a larger pore volume than the sheet NP-0. This indicates that not only the beating degree of the raw material fiber but also the drying condition changes the pore volume of the sheet. It can be seen that the pore volume of the sheet can be increased by drying without pressing. If this method is developed, it is expected to develop a cell culture bed apatite sheet having a larger pore volume if dry sheet formation is performed rather than wet sheet formation. However, the size of the hole is considered to depend on the cell type.

The characteristics of the apatite sheet as a culture bed cannot be judged only by the pore volume. The strength and air permeability of the sheet are also important at the time of actual use. These properties also depend on the beating degree of the raw pulp, and the tensile strength of the sheet NP-1 made from pulp with a high beating degree is large, but the sheet NP-0 is weak and cannot be said to be of practical strength. . On the other hand, the air permeability shows that the sheet NP-0 is small and has good air permeability. By using unbeaten pulp, it is possible to increase the pore volume to which cells adhere and at the same time reduce the air permeability that is involved in the permeability of the medium solution, but the strength is reduced. A sheet used as a culture bed needs to have a large pore volume, but at the same time, the sheet must be as strong as a filter paper because it is mainly used in a culture solution. In order to achieve both of these contradictory properties, the blending of binder fibers and the drying method without pressing become important. It was clarified that it is possible to produce a culture bed sheet having a large pore volume and good air permeability by the press-free drying method, and sufficient strength due to the strength enhancing effect of the binder.

Conventionally, a porous carrier has been used as a cell attachment material in cell culture in a spinner flask or the like. The cell-attached carrier is placed in a flask together with the medium, and suspension culture is performed while stirring at about 60 rpm with a stirring blade. Alternatively, the carrier is filled in a glass column or the like, and the carrier is cultivated while flowing by circulating the medium.
These methods are based on the Fluidized-bed method (Nilsso
n K., Buzsaky F. and Mosbach K., Bio / Technology,
4, 989 (1 986); Dean RCJr., Karkare SB, Ray
NG and Runstadler PWJr., "Continuous cell cult
ure with fluidized sponge beads: Large scale cell
culture technology ", Carl Hanser Vealag, NY, 145
(1987); Looby D. and Griffiths B., Trends Biotec
hnol., 8, 204 (1990)).

As a typical example of the carrier, Cellsnow (KIRI
N), Collagen-microsphere (Verax), etc. (KIRIN, animal cell culture carrier Cellsnow Text & Manua
l, 12 ； Ogawa T., Kamihira M., Terashima S., Yasud
a K., Iijima S. and Kobayashi T., J. Ferment. Bioe
ng., 74, 27 (1992); Shuji Terajima, Tatsuya Ogawa, Masamichi Kamihira, Kimiaki Yasuda, Shinji Iijima, Takeshi Kobayashi, Journal of Biotechnology, 71
(3), 165 (1993); Hayman EG, Ray NG, Runstad
ler PW JR, Bioreact. Biotransform., 132 (198
7)). Cellsnow is a porous particle made of cellulose with a size of about 2 mm and pores of 100 to 300 μm. Cel
In the CHO-K1 cell culture procedure using lsnow, the seed-cultured cells are inoculated into the carrier, and the concentration of the cell suspension at that time is 1.0 to 5.0 × 10 ⁵ cells / ml (KIRIN , Carriers for animal cell culture, Cellsnow Text & Manual, 12). 500m
Since 50 ml of Cellsnow is used in the l spinner flask, 50 to 250 × 10 ⁵ cells are required at the start of culture. This amount is 2 to 10 times the amount of cells when the apatite sheet of the cell culture bed is used as a culture bed in a 500 ml spinner flask. This is not only the case for Cellsnow, but also when using other porous carriers, a certain amount of cells or more is required (Hayman EG,
Ray NG, Runstadler PW JR, Bioreact. Biotran
sform., 132 (1987)). Because, when the carrier is fluidized and cultivated, each particle is liberated and the cell cannot grow into another particle. Therefore, a certain number or more of cells need to be attached to one carrier at the start of culturing, and the number of cells corresponding to the amount of carrier particles is required.
On the other hand, in the cell culture bed apatite sheet, the HAPC fiber surfaces to which the cells adhere are connected, and it is considered that the cells proliferate sequentially and spread over the entire surface of the sheet. Therefore, it is presumed that culturing is possible even when the initial cell concentration is relatively low.

Further, when the porous carrier is subjected to fluid culture, the carriers adhering to each other in the spinner flask or the column collide with each other and the growth of cells attached to the surface of the carrier occurs. On the other hand, since the cell culture bed apatite sheet is a single leaf sheet material, there is no contact between the materials and cell growth failure does not occur. Furthermore, since the sheet is fixed to the stirring blade, it is possible to easily replace all of the old culture medium by decantation. Since the porous carrier is suspended in the form of particles, it is impossible to completely exchange the medium.

The culture method in the spinner flask using the cell culture bed apatite sheet according to the present invention is a method which may be called a rotary bed (fluidized bed) culture method because the sheet is rotated together with the stirring blades. Is. In addition, compared to the conventional method, the number of cells at the beginning of culture is small, the immobilization procedure of the cells is simple, the growth disorder to the cells is less likely to occur during the culture, and the medium can be easily replaced. Due to such advantages, this culture method is the first excellent method in the world.

However, continuous culture by the batch method using the spinner flask according to the present invention seems to be limited in terms of medium supply. Therefore, we have developed a system that enables continuous supply of medium and continuous removal of waste products.

Perry SD et al. Proposed that the use of fiber materials as cell attachment materials has many advantages (Por
tner R., Ludemann I. And Markl H., Cytotechnology,
23, 39 (1997)). The advantage is the high S / V (Surface / Vol
ume) ratio, good separation of cells and medium, high medium permeability, low cost production of reactors, and easy scale-up. Then, they proposed a module in which glass fiber was packed in a column (Perry SD and Wang DIC, Biotechnol. B.
ioeng., 34, NO.1, 1 (1989)). It was possible to culture the CHO-K1 cells in a glass column reactor packed with 3.5 g of 80 μm diameter glass fiber at a higher density than the culture using microcarriers. However, this method is completely different from the method of using fibers in the form of sheets. Packing glass fibers in a column may not have been sufficient to take advantage of the fiber material. The proof is that the amount of attached cells was low. It has been confirmed from microscopic observation photographs that cells actually adhere to only a part of the glass fiber surface. In addition, it is considered that there is an imbalance in the growth of cells due to concentration gradients in the nutrient components in the medium at the entrance and exit of the glass column.

Next, two types of modules using non-woven fabric were studied (Mituda S., Matsuda Y., Itagaki Y., Su.
zuki A., Kumazawa E., Higa shio K. and Kawanishi
G., J. Ferment. Bioeng. ， 70，289 (1990); Robert
J., Cote J., and Archamsbault, J. Biotechnol. Bioe
ng. , 39, 697, (1992)). Among them, a non-woven sheet was used as the adhering material. A commercially available non-woven fabric whose raw material fiber is polyester fiber. Since polyester fibers have poor cell adhesion, plasma treatment is used to make them hydrophilic to improve cell adhesion, and sheets are used as they are. Since the diameter of the non-woven fiber used for each culture is only about 10 μm, it is too thin as a scaffold for cells, and as a result, the cell adhesion of the non-woven sheet does not seem to be sufficiently improved. . Further, in both studies, the supply of the culture solution is not a method of passing through the sheet, but a method of supplying the culture solution in parallel with the sheet surface. With this supply method, three-dimensional culture of cells cannot be expected because the culture solution is not sufficiently supplied to the inside of the nonwoven fabric.

A fixed bed type bioreactor using a non-woven fabric of very thick polyester fiber (diameter of 55 μm) which has improved those defects was proposed (Mo tobu M., Matsuo S., Wa.
ng PC, Kataoka H. and Matsumura M., J. Ferment. B
ioeng. , 83, 443 (1997)). A surface treatment was performed with type I collagen to improve the cell affinity properties of the polyester fibers. Since the fiber diameter was increased, the property of each fiber cell as a scaffold was improved. In addition, the growth of cells was improved because the medium was supplied through the sheet in a manner that allowed continuous culture for 41 days. However, since the morphology of the raw material fibers is columnar and the bulk density of the sheet is made too small, the voids between the fibers are too large and the extracellular matrix does not seem to be formed. The cell density per unit volume of sheet is 4.00 × 10 ⁶ cells / ml, which is smaller than 1.5 × 10 ⁸ cells / cm ^{3 of the} apatite sheet culture bed module.

Compared with the module using the above-mentioned fiber material, the following are the excellent features of the module using the apatite sheet. Since the surface is made of apatite, it has excellent cell adhesion without any surface treatment. Ribbon-shaped apatite-pulp composite fibers with a width of 60 μm are used as a raw material to form inter-fiber voids optimal for cell growth, resulting in a wide attachment area and high cell density for three-dimensional Cell can be cultivated. Furthermore, it is presumed that the distance between the fibers is appropriate, but it is easier for the cells to construct the extracellular matrix. A good growth condition is formed in the extracellular matrix constructed by the cells themselves inside the sheet. The cell culturing method using this module, such as the incorporation of cells into the reactor since the initial cell startup, is a very simple operation overall. The medium is fed through the sheet. Therefore, the cells proliferate and grow even inside the sheet, enabling cell culture in three dimensions. As a result, cells adhere to and grow on the entire sheet, enabling high-density cell culture.

[0042]

Example (Production Example 1) Synthesis of HAPC As a reagent for HAPC synthesis, calcium nitrate Ca (NO ₃ )
₂ (Wako Pure Chemical Industries, Ltd., Tokyo), Diammonium hydrogen phosphate (NH ₄ ) ₂ H (PO ₄ ) ₃ (Wako Pure Chemical Industries, Ltd., Tokyo), Sodium hydroxide NaOH (Wako Pure Chemical Industries, Ltd.) , Tokyo) was used. Commercially available bleached softwood and hardwood kraft pulp was used as the pulp.

As a result of examining various synthetic methods of HAp, HAP
C was synthesized by the wet method of HAp (Jarcho M., Bolen C.
H., Thomas MB, Bobick J., Kay JF and Doremus
RH, J. Materials Science, 11, 2027-2035 (197
6)) was utilized and it carried out as follows.

HAPC was synthesized by a method of dropping a diammonium hydrogen phosphate solution into a calcium nitrate solution by using an apatite wet synthesis apparatus (FIG. 1) according to the wet synthesis method of HAp. The reaction tank is a mini jar fermenter M-100 (Tokyo Science Instruments) used for culturing microorganisms etc.
Co., Ltd.) was used. The reaction tank is made of glass with a volume of 2 L and can be heated from room temperature to 60 ° C, and the solution in the tank is stirred up to 1500 rpm with a stirring blade using a magnet. Put 1 L of calcium nitrate solution in a reaction tank, add 15 g of absolutely dry pulp and 600 rpm
Stir at. When the pulp is completely disintegrated and the liquid temperature reaches 50 ° C, 300 mL of diammonium hydrogen phosphate solution is appropriately applied with a liquid feeding pump at a dropping rate of 5 mL / min. At the same time as the production of HAp, nitric acid is released in the reaction tank and the pH in the tank becomes acidic. Therefore, the reaction was proceeded while dropping 5N sodium hydroxide solution in order to maintain the predetermined pH. After dripping, HA
50 ℃ as a secondary aging reaction to promote the crystallization of p
After continuing stirring for 2 hours, the reaction was terminated.

In order to examine how the state of adherence of the synthesized HAp crystals to the pulp changes depending on the synthesis conditions (reaction temperature, pH of the reaction solution, etc.) and the type of pulp to be mixed, Table 1 The synthesis reaction was performed under the conditions.

[0046]

[Table 1] In Table 1, the synthetic amount of HAp is the amount of HAp synthesized from the calcium nitrate solution and the diammonium hydrogen phosphate solution in the table as raw materials. The symbols at the sample names are defined as follows to represent the reaction conditions. In this specification, the type of HAPC is represented by this notation. For example,
HAPC (N-8-1) uses NBKP as the raw pulp and has a temperature of 50
It was synthesized under conditions of ℃, pH8, HAp synthetic amount 6.02g. HAPC sample code: HAPC (PHQ) P: Kind of pulp (N: NBKP (softwood pulp), L: LBKP
(Hardwood pulp) H: pH of reaction solution (8, 9, or 10) Q: HAp synthesis amount (1 to 5) Describe the characterization of the complex.

(X-ray diffraction analysis of HAPC) HA attached to HAPC
A powder X-ray diffraction analysis of HAPC was performed to confirm p and to measure the amount of adhesion. An X-ray diffraction analysis of the apatite sheet was performed using a powder X-ray diffraction analyzer ABT-60 (Mac Science Co., Ltd.). As the X-ray source, a Cu wire generated at a voltage of 400 kV and a current of 100 mV was used. The HAPC processed into a sheet was used as a measurement sample, which was cut out according to the shape of the holder of the device and attached with double-sided tape for measurement.

The X-ray diffraction patterns of NBKP and N-9- (1-5) are shown in FIG. It can be confirmed that HAp is synthesized from the peak positions of the X-ray diffraction patterns of NBKP and HAPC. Since the peak shape is not sharp, it is considered that amorphous apatite is generated and attached.

Comparing HAPC under different synthesis conditions,
It can be seen that there is a difference in the peak intensity ratio of cellulose (2θ: 22.6 degrees) and HAp (2θ: 31.8 degrees). This indicates that there is a difference in the amount of HAp adhering to the pulp, suggesting that the adhering amount changes depending on the synthesis conditions.

Therefore, cellulose and HAp under each synthesis condition
The peak intensity ratio (Ι / Ι0) was calculated and plotted in Fig. 3. As is clear from the figure, the pH during synthesis
There is also a difference in intensity ratio depending on the amount of composition and the amount of composition. In particular, focusing on the amount of synthesis, it was found that there is a condition that the intensity ratio does not become larger as the amount of synthesis becomes larger, but becomes the maximum intensity ratio. N-8-4 was 87.6% under N-8- (1 to 5) synthesis conditions, and N-9-4 was 89.6% under N-9- (1 to 5) synthesis conditions.
% And the strength ratio is the maximum.

(HAp adhesion amount of HAPC) Each of the synthesized HAPCs was ashed at 850 ° C., and the ash content was measured as the weight of HAp, and the difference in the HAp adhesion amount due to the difference in the synthesis conditions was examined. The amount of H Ap attached per 1 g of pulp was calculated by the following formula. Adhesion amount of HAp (g / g) = ash content (g) / (fully dried HAPC (g) -ash content (g)).

Since it was found from the results of the X-ray diffraction spectrum that there was a difference in the adhered amount, the HAp adhered amount was measured.
The amount of HAp attached per 1 g of pulp at each synthetic amount is shown in FIG.

It was considered that the greater the amount of HAp synthesized, the greater the amount of HAp attached, but in reality it was found that there was a maximum amount of attached HAp. The tendency of the amount of adhesion was almost in agreement with the tendency of the X-ray intensity ratio of cellulose and HAp, and the samples of the maximum intensity ratio and the maximum amount of adhesion were the same, N-9-4.

When HAPC is synthesized by using NBKP as a raw material pulp, the following can be said as the tendency of the amount of HAp attached. p
Under the conditions of H8 and 9, the higher the synthesis amount, the higher the adhesion amount,
It became the maximum at -9-4, and the adhesion amount at that time was 0.57 g per 1 g of pulp. Under the condition of pH 10, even if the amount of synthesis increased, the amount of deposition did not increase so much, and it became the highest with N-10-3, and the amount of deposition at that time was 0.38 g per 1 g of pulp.

As mentioned above, the higher the pH of synthesis,
The higher the synthetic concentration, the more HA is attached to the surface.
It was observed that a large amount of p particles were attached. This phenomenon was observed in both NBKP and LBKP, and was determined by the concentration of the raw material liquid under HAPC synthesis conditions regardless of the pulp type.

As a result of synthesizing HAPC by setting various synthesis conditions such as the type of pulp or the pH and concentration of the reaction liquid, it became clear that the amount of HAp deposited on the HAPC surface changes, and the conditions can also be grasped. (See FIG. 4). Although data are not shown, 0.72 g of HAp was attached to 1 g of pulp in L-10-4. From these results, by setting a certain synthesis condition, the amount of HA Ap attached to HAPC per 1g of maximum pulp
It can be adjusted up to 0.72g. Although it is not clear why there is an optimum amount, it is possible that the ion adsorption reaction between the carboxyl groups on the pulp surface and the calcium ion that forms the crystal nucleus of HAp, and the effect of the ion concentration of the reaction solution. Furthermore, it is speculated that various factors such as packing of HAp particles in the pulp lumen and crystallization of HAp particles in the cell wall are involved in this reaction.

(Observation of HAPC surface) The condition of the surface of HAPC and the change in the shape of HAp particles adhering to the pulp surface due to the difference in the synthesis conditions were examined using a scanning electron microscope (hereinafter abbreviated as SEM). After air-drying the sheet, ion sputter (E-
1030, Platinum-palladium (Pt-P) by Hitachi, Ltd.
d) Deposition was performed and observed using SEM (S-4500, Hitachi, Ltd.). Fig. 5 shows a photograph of the surface of HAPC (N-8-1, N-8-4) observed with an electron microscope.

Fine cellulose fiber lines are clearly observed on the surface of NBKP. Furthermore, the shape of the holes leading into the lumen of the pulp surface is clear. However,
Looking at the HAPC surface, it can be seen that the HAp particles cover the pulp surface as if cement had been applied. In N-8-1, the shape of holes on the pulp fiber surface is observed, but in N-8-4, the HAp is so thickly laminated that the hole shape cannot be observed. In other words, it can be said that HAPC has a sheath / core structure in which pulp is used as the core and HAp is covered around it. Organic pulp and inorganic
It is an organic-inorganic composite fiber material in which HAp is composited. Such a fibrous material has never been manufactured and is a novel material.

(Composite technology of pulp fiber and inorganic powder) HA
PC can also be regarded as pulp with the function modified by modifying the pulp surface with apatite. Table 2 shows a comparison of the properties of the functional pulp, which was created by combining the inorganic powder and the pulp that have been developed so far, with the properties of the apatite-pulp composite.
It was shown to.

[0060]

[Table 2] Similar to the method of combining pulp and inorganic powder for use like HAPC, Scallan et al. Or Miller
Et al., Method for compounding titanium oxide (Green HV, Fox
TJ and Scallan AM, Pulp & Paper Canada, 8
3, 7, T203 (1982); Middleton SR and Scallan A.
M., Colloids and Surfaces, 16, 309-322 (1985); Scal
lan AM and Middleton SR, Papermaking Raw Mat
erials, Fundamental Reseach Symposium (8th) Oxfor
d, Vol.2, 613 (1987); Miller ML and Paliwal D.
C., J. Pulp Pap. Sci., 11, NO.3, J84 (1985)), and Allan et al.'S method of complexing calcium carbonate (Alla
n GG, Carroll JP, Negri AR, Raghuraman
M., Ritzenthaler P. and Yahiaoui A., Tappi J., 175
(1992); Allan GG, Carroll JP, Negri AR, R
itzenthaler P. and Yahiaoui A., Tappi J., 239 (199
2); Allan GG, Ko YC and Ritzenthale rP., Tap
pi J., 205 (1991)). In these methods, pulp having no drying history is used as a raw material, and an inorganic powder is attached to the inner cavity of the pulp and the pores of the pulp cell wall by a physical method, a chemical method, or a method using a fixing agent,
This is a method that can be called an inorganic powder composite pulp method in which inorganic powder composite pulp is created and made into paper.

Scallan et al. Prepared an inorganic powder composite pulp as follows. First, 15 g of softwood chemical pulp, which has no drying history, is put into 1.25 L of deionized water for disaggregation. Separately, 30 g of titanium oxide is dispersed in 250 ml of deionized water and added to the pulp dispersion. This dispersion is strongly stirred at 3,000 rpm for 20 minutes, and titanium oxide is put into the pulp. Next, this was transferred to a washing device and washed until the washing water became clear, and the inorganic filler adhering to the pulp surface was removed to prepare titanium oxide composite pulp. Aluminum sulfate is used to fill the pulp with titanium oxide. As the added amount of aluminum sulfate increases, the surface potential of titanium oxide becomes positive and the filling amount of the lumen increases. Comparing the bore fillings of softwood pulp and hardwood pulp, softwood pulp was filled with 10 to 13 wt% of pulp with no drying history, and hardwood pulp had a very low filling volume of 2 to 3 wt%, probably because of its small lumen volume. It was It is considered that the addition of aluminum positively changed the potential on the titanium surface, making it easier to electrostatically combine with the negative charge of cellulose. From these things, Scalla
N et al. propose that the powder adheres by electrostatic force and van der Waals force.

Allan et al. Filled the pores of the cell wall with an inorganic powder such as calcium carbonate by an in situ chemical reaction method. They used red alder bleached kraft pulp with no drying history. 6 g of pulp is treated with an aqueous solution of calcium chloride (10 to 120
g / L) for 5 minutes, scoop up with a 100 mesh wire to obtain a pulp concentration of about 8%. Next, this was put into an aqueous solution of sodium carbonate (10 to 120 g / L) and subjected to an in situ chemical reaction to generate calcium carbonate in the pores. The complexed pulp after the reaction was thoroughly washed with water to remove the powder adhering to the surface. As a result, we succeeded in depositing up to 18% of calcium carbonate.

Their purpose was to increase the proportion of the inorganic additive, which is relatively cheaper than pulp, in the paper in order to reduce the manufacturing cost of the paper product. Simply increasing the amount of inorganic additives causes the powder to adhere to the pulp surface, resulting in a decrease in paper strength. To prevent this, pulp with no history of drying is used to fill inorganic powder into the space where the strength of the paper is unlikely to decrease, such as the lumen. The advantage of the inorganic powder in the pulp cavity is that the inorganic powder is protected by the pulp cell wall during the papermaking process and does not escape to the outside. It is said that the bond between pulp fibers is stronger than in the method. They did not pay much attention to the adhesion of powder to the pulp surface because the strength of the paper decreases, and they considered to produce a composite that minimizes the amount of inorganic powder adhering to the pulp surface. ing.

Apart from these studies, Sakata et al. ([37,38
37] Sakata Isao, Kenji Takeshita: Patent Gazette Sho 62-4414; Isao Sakata,
Kenji Takeshita: Patent Publication Sho 62-62187), Marchessault (Ma
rchessault RH, Rioux P. and Raymond L, Polymer,
33, NO.19, 4024 (1992); Marchessault RH, Richar
d S. and Rioux P., Carbohydr. Res., 224, 133 (199
2); Rioux P., Richard S. and Marchessault RH,
J. Pulp Pap. Sci., 18, NO.1, J39 (1992)) and Fujiwara et al. (Katsuhiro Fujiwara, Journal of Paper and Paper Technology, 47, 437 (1993)). Sakata et al. Have developed a method for producing magnetic pulp by pre-activating the pulp and then treating it with a metal complex salt solution. As the activation treatment, a reducing functional group such as an aldehyde group is introduced into the pulp by oxidation treatment with an oxidizing agent such as periodate, and the reducing property is exerted to precipitate the magnetic metal on the pulp surface or inside, Create a magnetic pulp. In the method of Sakata et al., A functional group is chemically introduced in advance, so that the type of pulp may be either dry pulp or pulp with no drying history. By converting this pulp into sheets, it is possible to make paper that can be used for magnetic cards or sheets that have an electromagnetic wave shielding function.

Marchesault et al. Filled the magnetic powder in the pulp cavity by vigorously stirring the commercially available magnetic powder and the softwood pulp. Disperse 15 g of pulp in 1,250 ml of pure water. Disperse 15 to 45 g of magnetic powder (magnetite) in 250 ml of pure water. The pulp liquid and the magnetic powder dispersion were mixed, aluminum sulfate was further added, and the mixture was vigorously stirred at 3,000 rpm for a predetermined time, and titanium oxide was put into the pulp cavity from the pulp pit. After filling the lumen, cationic polyethyleneimine was added as a retention aid. The magnetic powder adhering to the pulp surface was washed and removed in running water. 20g / of magnetic powder
It was found that if the concentration was L or higher, 10 to 20 wt% of the lumen could be filled in about 20 minutes at 3,000 rpm. A magnetic sheet is produced by making this pulp into a sheet. It is said that a polymer fixing agent is necessary to prevent the magnetite from falling out of the pulp lumen during the sheeting process in the papermaking method.

Fujiwara et al. Produced a magnetic powder composite pulp by performing an in situ reaction in the pulp lumen. Pulp with no drying history is dispersed in an aqueous solution of ferrous salt, etc., and ferrous ions, etc. are put into the pulp cavity via the pulp pit. An alkali is added to this to produce ferrous hydroxide and the like.
Next, this reaction solution is maintained at a predetermined temperature and air is blown into it to carry out an oxidation reaction for a predetermined time. After completion of the reaction, the magnetic powder composite pulp is washed in running water to remove the magnetic powder on the pulp surface. It was confirmed that the magnetic powder generated in the pulp lumen was magnetite and manganese ferrite. A large amount of magnetic powder, not a single layer, on the surface of the pulp lumen, up to 45
wt% loaded.

It can be said that the HAPC production technology has the following features as compared with the production technology of these inorganic powder composite pulps. First, the HAPC manufacturing method is a chemical method because it is a method of precipitating insoluble inorganic salts. Another feature is that HAp, which is an inorganic powder, adheres to the pulp in a larger amount than the physical method, as in other chemical methods. Apatite-adhered pulp can be prepared by physical methods, but its HAp adhesion amount is 1
It is 0 to 17 wt%, which is not different from the amount of titanium oxide deposited by the strong stirring method.

FIG. 6 shows the difference in the amount of HAp attached due to the apatite attached pulp by the strong stirring method using NBKP (softwood pulp) having a drying history as the raw material fiber and the HAPC synthesis reaction. The HAPC method corresponds to the synthesis condition of N-8- (1-5), and in the strong stirring method, 15 g of pulp was added to the HAp reaction solution synthesized without pulp under the condition of N-8 and strong stirring was performed. did. As is clear from the figure, as the amount of HAp to pulp increased with the strong stirring method, the amount of HAp deposited also increased, but the maximum was 17 wt%. However, aluminum sulfate was not added in this strong stirring method. Further data is not shown, but LB with a history of drying
It was found that 10 to 15 wt% of HAp was attached even with KP.
This is because the lumen of hardwood with a history of drying has a small volume and is filled with only 2 to 3 wt% of titanium oxide (Green HV, Fox TJ and Scallan AM, Pul.
p & Paper Canada, 83, 7, T203 (1982); Middleton
SRand Scallan AM, Colloids and Surfaces, 1
6, 309-322, (1985)). The fact that 10 to 15 wt% of HAp adheres to both softwood pulp and hardwood pulp with a history of drying indicates that HAp adheres to the pulp surface by the strong stirring method.

On the other hand, in the HAPC method, the adhesion amount is 20 wt% even under the condition of 6/15 (condition of N-8-1). Furthermore, as the amount of HAp synthesized increases, the amount of HAp deposited also increases, and N-8-4 deposits about 2.5 times the amount deposited by the strong stirring method under the same conditions. In the HAPC method, the reaction proceeds while dropping the reaction solution over time. As a result, crystallization of HAp on the pulp surface is repeated little by little, and crystallization adhesion progresses further on the first HAp layer, so it is considered that a large amount of HAp adheres. . In the strong stirring method, since the already synthesized Hap particles are physically attached, it is considered that such a phenomenon does not occur and only a certain amount is attached.

[0070] Considering that the application of the powder fixed on the pulp surface to the ion adsorption function of the powder will be expanded to include biocomponent adsorption sheets, there is an inorganic powder having a function like HAPC on the fiber surface. It is better to have been doing. Furthermore, for the purpose of adhering to the pulp surface, it is not necessary to use pulp that has no drying history, and commercially available dry pulp can be used as a raw material for complexing with HAp. Also, H
Since the APC synthesis method utilizes the insoluble salt formation reaction of HAp, so-called functional pulp can be produced by omitting the chemical operation of previously introducing a functional group into pulp.

As described above, an apatite-pulp composite (HAPC) in which HAp is crystallized and attached to the surface can be prepared by mixing pulp during the wet synthesis reaction of HAp. This synthetic reaction is a method in which HAp, which is an insoluble inorganic salt, crystallizes from pulp fibers as the nucleus.
It was found that the amount of adhesion can be adjusted arbitrarily by changing the synthesis conditions. HAPC uses pulp as the core and HA around it.
It is a fiber with a sheath / core structure covered by p and can be restated as an organic / inorganic composite fiber material. Since the surface of the fiber contains only HAp, it may be called HAp fiber, and as a material form that has been difficult to use until now, for example, a sheet material with HAp ion adsorption characteristics and biocompatibility characteristics. Available.

Example 2 A method of forming HAPC on an apatite sheet will be described.

As described above, HAPC is a functional fiber material that is a composite of pulp and HAp. Since pulp is the raw material, HAPC has the same morphology as pulp fiber. Therefore, the paper-making method, which is a method of making paper, can be easily made into a sheet, and an apatite sheet can be made (Kawakat
su H. and Ide S., Okuda Y., Sen'I Gakkaishi, (200
0)). Since the surface of this apatite sheet is HAp, it is a functional sheet material that is given its characteristics of ion adsorption and biocompatibility. Apatite sheet exhibits excellent performance as an adsorbent for biological components such as proteins and nucleic acids, or as a nucleic acid separation and fractionation sheet (Kawak
atsu H. and Ide S., Okuda Y., Sen'I Gakkaishi, (200
0)).

(Manufacturing Method of Sheet) As samples, four kinds of HAPC (NP-1, NP-2, LP-1 and LP-2) were synthesized under the synthesis conditions shown in Table 3 according to the above HAPC synthesis method. . The raw material pulp is commercially available bleached softwood kraft pulp (NBKP),
Bleached hardwood kraft pulp (L-BKP) was used. The HAPC synthesis conditions are shown in the table below.

[0075]

[Table 3] The HAPC suspension synthesized above was divided into four equal parts, and using this as a raw material fiber, four apatite sheets were prepared in accordance with JIS P8209 (Handmade paper adjustment method for pulp test). However, the wet paper is made using a square sheet machine of 25 × 25 cm ² (Tester Sangyo Co., Ltd.), and the wet paper is a cylindrical electric heater type rotary dryer (Kumagaya Riki Kogyo Co., Ltd.). And dried at 120 ° C. A sheet having a pulp basis weight of 60 g / m ² was prepared.

(Characteristic Evaluation of Sheet) Observation of HAPC in the sheet, the state of apatite on the surface, entanglement between fibers, and the like were performed using SEM. After air-drying the sheet, platinum-palladium vapor deposition was performed by ion sputtering (E-1030, Hitachi, Ltd.), and observed using SEM (S-4500, Hitachi, Ltd.).

To confirm the apatite contained in the apatite sheet, a powder X-ray diffraction analyzer (ABT-60, manufactured by Co., Ltd.) was used.
X-ray diffraction analysis of the apatite sheet was performed using Mac Science. As the X-ray source, a Cu wire generated at a voltage of 400 kV and a current of 100 mV was used. The trial-produced sheet was cut as it was into a sample, cut into a required size, set in a sample holder, and measured.

In order to determine the amount of apatite contained in the sheet, the sheet was ashed at 850 ° C., the ash content was measured, and the weight% of ash content per sheet was calculated as the apatite adhesion amount.

To examine the strength characteristics of the sheet, JIS P811
3 (Test method for tensile strength of paper and paperboard) and JIS P8116
The tensile strength and tear strength of the apatite sheet were measured according to (Test method for tear strength of paper and board).

(Protein / Nucleic Acid Adsorption Ability of Sheet) In order to examine the adsorption property of biological components of the apatite sheet, the adsorbed amount of protein and nucleic acid on the sheet was measured. Protein is BSA (bovine serum albumin), egg white lysozyme (Wako Pure Chemical Industries, Ltd.), nucleic acid is DNA (salmon sperm), RNA
Two types (derived from yeast) (Wako Pure Chemical Industries, Ltd.) were used.
1/15 mol / L phosphate buffer solution (Phosphate Buffer hereafter, abbreviated as PB) containing about 500 mg / L of each component (pH 6.8)
(Wako Pure Chemical Industries, Ltd.) (However, BSA was 1/60 mol / L PB) 50 ml and apatite sheet 0.5 g were put in a glass sample bottle, and after 48 hours, the absorbance of the solution was measured using a spectrophotometer. The amount of each component adsorbed was determined from the difference in absorbance with the blank test solution without the sheet. The measurement wavelength is for proteins
The wavelength was 280 nm and the nucleic acid was 260 nm.

(Characteristics of Apatite Sheet Manufacturing Method) HA
Four kinds of apatite sheets were prepared by the papermaking method using PC as the raw material fiber. The basis weight of the sheet is 60 g / m ² as the basis weight of pulp alone, but due to the apatite adhering to it, a sheet of 75 to 100 g / m ² was actually obtained. Since the apatite is a white inorganic powder, the created apatite sheet looks white like a filter paper for chemical analysis. Four types of sheets are available, sheet NP-1, sheet NP-2, and sheet L, depending on the type of HAPC used as the raw material (Table 4).
Referred to as P-1 and sheet LP-2. Although the paper was made using a square sheet machine, it could be easily made into a sheet without problems such as fiber aggregation. Even without using a dispersant, the dispersibility of HAPC in the vat of the sheet machine was good. The wet paper web after sheet formation was easy to handle, and there was no problem in transferring it to the drying process.

[0082]

[Table 4] The above-mentioned apatite sheet production method using HAPC as the raw material is referred to as the HAPC method here, and the conventional inorganic powder paper production method (Lindstroem T., Kolseth P. and Naeslund
P., Pap. Raw. Mater., 2, 589 (1985); Lindstroem
T. and Kolseth P., Paper, 200, NO.9, 34 (1983); Katsumi Kuboshima, Research Paper on Functional Paper, 21, 31 (1982); Katsumi Kuboshima, Toshihiko Kurata, Research Paper on Functional Paper, 23, 22 (1984)) and explain the characteristics of the "HAPC method".

The method for producing an apatite sheet by the HAPC method is as shown in FIG. It is an excellent method compared to.

When the apatite sheet is produced by the conventional method, it is necessary to dry and pulverize the synthesized apatite in order to make it into the form of fine particles which are the raw material for the sheet, which takes a lot of labor and time. On the other hand, in the HAPC method, the process can be omitted and the method is excellent in cost. In the conventional method, a polymeric fixing agent or the like is required to infuse HAp into the sheet, and it is necessary to take a method of forming an aggregate of HAp and pulp by the fixing agent and forming the sheet into a sheet. Further, in this method, a polymer fixing agent forms a film on the surface of the apatite, which causes a problem that the ion adsorption property and biocompatibility property of the apatite are reduced. Since the HAPC method does not use a fixing agent, such a problem does not occur. The HAPC method is a new apatite sheet manufacturing method that can solve these problems in conventional inorganic powder paper manufacturing methods.

Since the most important synthesis reaction of the apatite-pulp composite is also an aqueous reaction, it was possible to develop a synthesis device by improving pulper, which is a paper manufacturing facility well known to companies. In addition, using the newly developed synthesizer, HAPC
Was synthesized, and finally an apatite sheet was created.

(Characteristics of Sheet) FIG. 8 shows an SEM observation image of the apatite sheet (NP-1, LP-1). It can be confirmed that the apatite layer is laminated on the surface of the pulp fiber in each of the apatite sheets. Sheet NP-1 and sheet LP-1 are
Although the amount of adhesion is small, it is observed that it adheres to the pulp surface. Sheet NP-2 and sheet LP-2 seem to be distinctly different from the pulp surface, and apatite is attached as a thick layer. In addition, a small amount of apatite particles that did not adhere to the pulp fibers remained between the fibers. However, most of the apatite contained in the sheet is HAPC.
It is considered that the particles are apatite particles that have been composited with, and the particles did not fall off from the dried sheet.
This is completely different from the fixing state of the powder in the paper in which the inorganic powder is blended by the internal spinning method. For example, talc with coarse particles (particle diameter 2 to 10 μm) exists in the dents on the paper surface and in the spaces between fibers. On the other hand, aluminum hydroxide with fine particles (particle size 0.5 to 1 μm) is agglomerated and fixed on the fiber surface rather than in the interstices between the fibers,
Instead of coating the entire surface of the pulp, it covers a part of the pulp (Tadahei Hamada, Paper Science, Chugai Industrial Research Society (existing), 424 (1982)). For this reason, there is a problem in that the inorganic powder paper made by internally adding the inorganic powder to the pulp is apt to fall off the powder (Koshitoshi Sugiyama, Functional Paper Research Paper, 37, 13 (1998)). It is shown that the apatite sheet produced by the HAPC method is a sheet formed by combining and integrating the apatite particles with the pulp, unlike the conventional inner liner paper.

FIG. 9 shows X-ray diffraction patterns of the apatite sheet and the pulp sheet. From the X-ray diffraction pattern of the apatite sheet, a peak of 2θ = 15 to 20 degrees derived from pulp (cellulose) and characteristic peaks of apatite of 26 degrees and 31 to 32 degrees were confirmed. From the ratio of the peak intensity of pulp to that of apatite, it can be seen that the amount of apatite deposited on sheet NP-2 and sheet LP-2 is about twice that on sheet NP-1 and sheet LP-1. In fact, the apatite content weight% calculated from the ash content of the sheet was about twice as large (Table 5).

[0088]

[Table 5] The apatite weight% of the sheet and the strength characteristics are shown in Table 5. When the recovery rate of the apatite synthesized from the apatite weight% was calculated, it was found that the theoretical amounts of the sheets NP-1 and LP-1 were 64 and 70%, respectively.

As is clear from the table, the weight% apatite in the sheets NP-2 and LP-2 doubled to 39.8 and 36.8%. This means that 49,42% of the theoretical HAp synthetic amount was recovered. Sheet NP-2, which is a high-concentration apatite synthesis condition
Also, when evaluated from the perspective of recovering the apatite synthesized from the sheet LP-2, less than 50% was recovered, resulting in poor recovery efficiency.

Regarding the sheet strength, the strength of the sheet NP, which is a raw material fiber of pulp having a relatively long fiber length, is higher than that of the sheet LP having a short fiber length. Higher strength than those with many sheets with low apatite content. The tensile strength of the sheet NP is higher than that of the commercially available filter paper 51B. The tear strength of sheet LP is less than half that of sheet NP, which is lower than that of filter paper 51B. However, even the LP-2 sheet, which has the lowest strength, can be used even when it contains water, as with the filter paper 51B, with a little care. In addition, in general, the sheet strength of the sheet in which the inorganic powder was mixed by the internal addition method was significantly decreased when the powder content was increased, but it was revealed that the HAPC method can reduce the decrease of the sheet strength ( Kawakat
su H. and Ide S., Sen'i Gakkai Prepr. P-5 (199
8)).

How much apatite should be contained in the apatite sheet depends on the application of the sheet. Therefore, it is necessary to control the amount of apatite deposited by changing the HAPC synthesis conditions. With the HAPC method, it has become possible to manufacture apatite sheets while taking into consideration the use of the sheet, the required amount of apatite adhered to it, the strength characteristics of the sheet, etc. ， 28 (199
3)). Needless to say, determining the amount of adherence also affects the manufacturing cost of the sheet, because the recovery rate also changes when the amount of adherence of apatite changes.

(Sheet Protein / Nucleic Acid Adsorption) FIG. 10 shows the amount of protein / nucleic acid adsorbed on the apatite sheet. In FIG. 10, the concentration of 50 ml solution of each component is
BSA 480 mg / L, lysozyme 550 mg / L, D
NA is 500 mg / L, RNA is 520 mg / L, and the solvent concentration is BSA, 1/60 mol / L, pH 6.8.
Phosphate buffer (PB), lysozyme, DNA, and DNA were 1/15 mol / L and pH 6.8 PB, respectively. As is clear from the figure, sheets NP-1 and LP-1, or sheets NP-2 and LP-, which have almost the same apatite content,
There was almost no difference in the amount of protein / nucleic acid adsorbed between the two. When comparing the fiber sizes of NBKP and LBKP, the former is
Although it is about three times as large, it means that there is almost no difference in the adsorption amount due to the difference in the form of HAPC fibers that are the raw material of the sheet. However, comparing the sheets NP-1 and NP-2, which differ in the apatite content by about 2 times,
Nearly twice as much is adsorbed. As a comparison, the adsorbed amount of the commercially available filter paper 51B was measured, but neither protein nor nucleic acid was adsorbed. Since it is apatite that exhibits the adsorption property, it is natural that the adsorption amount varies depending on the content. Sheet protein
It was revealed that the adsorption property of nucleic acid depends on the content of apatite in the sheet and is not affected by the fiber morphology of pulp. The amount of protein adsorbed on the apatite sheet LP-2 was 13 mg / g of BSA per sheet weight, 7.9 mg / g of lysozyme, and the amount of adsorbed nucleic acid was 4.6 mg / g of DNA and RNA.
It was 5.7 mg / g. This amount is a commercially available membrane sheet for protein / nucleic acid hybridization (eg, nitrocellulose membrane (Bio-Rad.
Laboratories Life Science Division General Catalog
1998/99, 308; Hisashi Hirano, Protein structural analysis for gene cloning, Tokyo Kagaku Dojin, 45 (199)
3); Matsudaira P., J. Biol. Chem., 262, 10035 (19
87)) Adsorption amount is about the same. In addition, the size of several tens of μ produced by granulating and sintering apatite currently in practical use.
Hydroxyapatite chromatography carrier ([5353] Bio-Rad Laboratories.
Life Science Division General Catalog 1998/99, 103)
Both have the same adsorption performance.

(Example 3) In this example, an example in which the apatite sheet is used as a cell culture bed apatite sheet will be described.

As a sample, a 0.06 mol / L calcium nitrate solution in which 15 g of unbeaten commercial bleached softwood kraft pulp (NBKP) was suspended using a HAPC synthesizer according to the above method
300 ml of 0.12 mol / L diammonium hydrogen phosphate solution in 1000 ml
HAPC was synthesized by the method of dropping l. HAPC (sample name is abbreviated as HAPC (NP-0)) was prepared while controlling the pH using a 5 mol / L sodium hydroxide solution at a reaction temperature of 50 ° C. and a reaction pH of 8 as the reaction conditions. It has been confirmed by ash weight measurement that HAPC (NP-0) has 20 wt% of apatite attached.

(Preparation of cell culture bed apatite sheet) HA
Using PC (NP-0) as a raw material, the apatite sheet was prepared by drying at 120 ° C. using a square sheet machine and a cylindrical electric heater type rotary dryer according to the above-mentioned sheet manufacturing method. By this operation, most of the unattached apatite particles in the HAPC (NP-0) suspension are removed as a sheet machine filtrate. However, since the apatite particles that were not slightly complexed remained in the sheet, the sheet was disintegrated again with a stirrer to remove it, and this was used as a raw material for the cell culture bed apatite sheet HAPC (NP -0) made into a suspension. 80 wt% of raw material HAPC (N P-0) and 20 wt% of EKC (Chisso Polypro Fiber Co., Ltd.), a fibrous binder of polyolefin type, were mixed, and after paper making with a square sheet machine, natural convection drying without press dehydration. It was dried in the machine. At that time, the drying temperature is higher than the melting point of the binder (105-130 ℃).
℃ was made. Since the rotary dryer used for press-drying conventional apatite sheets is not used, a sheet material with less inter-fiber adhesion and wide voids is created. as a result,
Basis weight 85g / m ² and 175g / m ² with low bulk density and large porosity
Two kinds of cell culture bed apatite sheets having a size of 25 × 25 cm ² were obtained. Abbreviated as HAp-SB (85) and HAp-SB (175), respectively.

(Characteristic Evaluation of Cell Culture Bed Apatite Sheet) After air-drying the cell culture bed apatite sheet, Pt-Pd deposition by ion sputtering (E-1030, Hitachi, Ltd.) was performed, and then SEM (S-4500). , Hitachi, Ltd.) was used to observe the sheet surface. Also, a mercury porosimeter (Porasizer 9310, Shimadzu Corporation)
The pore volume and surface area of the pores present in the sheet were measured by.

(Static Culture Using Cell Culture Bed Apatite Sheet) CHO-K1 cells (Chinese hamster ovary cells) (RCB0285, RIKEN Genebank / Cell Development Bank) were used for the culture experiment. For the initial culture, Ham's F-12 (Gibco BRL) medium containing 10% fetal bovine serum (FBS) (Gibco BRL) was used, and carbon dioxide concentration was maintained in a carbon dioxide incubator.
The culture was performed in an atmosphere of 5% air and 95%. A 0.25% trypsin solution (containing 1 mM EDTA) (Gibco BRL) was used as a cell dispersant at the time of cell passage. HAp-SB (85) was washed twice with distilled water, autoclaved in distilled water at 121 ° C for 20 minutes, and then washed twice with culture medium.

Confluent cells that had been passaged several times were placed in a 60 mm plastic culture dish to obtain 25 × 25 mm ² H ² H ² .
2.8 × 10 ⁵ cells were inoculated on Ap-SB (85) and 7 ml of Ham's F-12 medium was added. The medium was replaced every 2 days in the carbon dioxide incubator and cultured for 9 days. The medium was exchanged by transferring the sheet to which the cells were attached to a new 60 mm plastic culture dish containing 7 ml of fresh medium by picking with forceps.

After completion of the culture, the morphology of CHO-K1 cells adhered and grown on the surface of the apatite sheet was observed by using SEM.
The cell-attached sheet was lightly washed with PBS (Gibco BRL), dehydrated with an ethanol increasing series (70, 80, 90, 95, and 100%), and finally replaced with t-butyl alcohol, followed by a freeze dryer. After drying with (ES-2030, Hitachi, Ltd.), Pt-Pd vapor deposition by ion sputtering (E-1030, Hitachi, Ltd.) is performed and SEM (S-4500, Hitachi, Ltd.) is used. And then observed.

The sheet to which the CHO-K1 cells were attached was taken out from the culture dish, washed with 40 ml PBS, and immersed in 7 ml of a 0.25% trypsin solution (containing 1 mM EDTA) for 1 minute. Then 10
The cells were removed from the sheet by sufficiently washing in 30 ml of Ham's F-12 medium containing% FBS. The detached cells were stained with Trypan blue staining solution, and the number of cells was counted using a hemocytometer and a phase contrast microscope.

(Continuous Culture in Spinner Flask) HAp-
A continuous culture experiment of CHO-K1 cells in a spinner flask (Bellco) for a 500 ml microcarrier (FIG. 11) using SB (175) was performed by the following method.

The cells used for continuous culture (the above-mentioned CHO-K1 cells) were Ham's F-12 medium containing 10% FBS and 5% carbon dioxide gas.
/ Pre-incubated at 37 ° C in a 95% carbon dioxide incubator. 0.25% as a cell dispersant for cell passage
A trypsin solution (containing 1 mM EDTA) was used. The sheet used for culturing was previously washed twice with distilled water in a state of being set on a stirring blade, sterilized by autoclave, and then washed twice with a medium. A sheet (size (width, length) 35 x that was set up so that confluent cells were subcultured several times in a culture dish and wrapped around a stirring blade in a flask.
3.0 × 10 ⁶ cells were inoculated on 410 mm, weight 3 g) and cultured. Since the culture bed is in the form of a flexible sheet, it can be set around the stirring blade (Fig. 11). This method is a novel flask culture method that is completely different from the culture methods reported so far.

[0103] 2 of size 25 × 25 cm ² by (cell culture properties of the cell attachment surface of the floor apatite sheet) Sheet create operation
Types of cell culture bed apatite sheets (HAp-SB (85) and HA
p-SB (175)) was obtained. The main characteristics of this sheet are shown in Table 6. As a comparison, the published method (Bernardi G., Me t
hods of Enzymology, XXI, Academic Press, (WB Jakob
y, Ed.) New York, 95 (1971)) based on HAPC (NP-0)
Apatite sheet (abbreviated as sheet NP-0)
And HAP made from NBKP adjusted to a beating degree csf of 420 ml
An apatite sheet (sheet NP-1
Abbreviation) was created. It has been confirmed by X-ray diffraction spectrum that amorphous apatite adheres to these sheets (Hironobu Kawakatsu, Research Paper of Functional Paper, 32, 28 (1
993)). FIG. 12 shows the pore volume distributions of the three types of sheets. Table 7 shows the results in which the pore volume, surface area, fracture length and air permeability of the cell culture bed apatite sheet obtained from the pore volume distribution are summarized.

[0104]

[Table 6]

[0105]

[Table 7] An electron microscopic observation image of the cell culture bed apatite sheet HAp-SB (85) is shown in FIG. Approximately 200 width from observation image to sheet
It is confirmed that there are many gaps of less than μm. Although it is measured as pores in the porosimeter analysis, it is observed that it is a gap between fibers. The feature of the gap of this sheet is that the sheet is made by laminating HAPC monofilaments by the papermaking method, so that the gap is connected from one side (front) to the other side (back). Is mentioned.
Considering the performance as a cell culture bed, it is important that cells adhere to and grow inside the sheet and are cultured at a high density. For that purpose, the culture solution must be efficiently flowed into the culture bed to supply nutrients and oxygen. For a good flow, it is necessary for the culture bed to have holes of a certain size connected continuously to the inside.

The cell growth curve on the surface of HAp-SB (85) in the culture dish is shown in FIG. The number of cells on the surface of the sheet was 3.0 × 10 ⁶ cells / sheet on the 4th day of culture, and 9.0 × 10 ⁶ cells on the 7th day of culture.
It became a piece / sheet. After that, the number of cells did not increase and the cell density became saturated. When the number of cells per unit area is calculated based on the area of the sheet, it is 1.5 × 10 ⁶ cells / cm ² , and the cell density in a commonly used plastic culture dish is 1.1 × 10 ⁵ cells / cm ² . is there. It was clarified that the use of the culture bed apatite sheet enables the culture to be performed at a cell density about 10 times higher than that of the culture dish. So far, glass fiber woven fabrics and porous polytetrafluoroethylene membranes have been investigated as immobilization carriers for CHO-K1 cells. Among them, the cell density of glass fiber woven cloth was 3 × 10 ⁵ cells / cm ² and that of porous polytetrafluoroethylene membrane was 8 × 10 ⁵ cells / cm ² , which is a promising material as an attachment carrier. It has been done (Junko Kano, Takayoshi Tokiwa, Meng X., Ryo Kodama,
Matsumura Soushi, Biomaterials, 15, NO.2, 55 (1997); Tokiwa
T., Kano J., Kodama M. and Matsumura T., Cytotechno
logy, 25, 137 (1997)). Compared with these materials, the cell culture bed apatite sheet can achieve a cell density of 2 to 50 times, and it can be said that it is a material with excellent cell adhesion and cell proliferation. This property is considered to be due to the fact that the apatite sheet has an apatite surface with excellent biocompatibility and that the fiber is a sheet-shaped porous material having a large surface area.

Further, since this culture bed is in the form of a sheet, it can be easily moved, and it can be replaced with a fresh medium simply by grabbing the sheet with a pair of tweezers and transferring it to a culture dish containing a new medium. Was also found.

The medium used for continuous culture was 10% FBS and 20 mM.
450 ml Ham's F-12 containing HEPES buffer (Gibco BRL) was used. The flask was placed in a constant temperature bath at 37 ° C., and closed system culture was performed while rotating the stirring blade around which HAp-SB (175) was wound at 60 rpm. Since the sheet is set on the stirring blade, the sheet itself rotates together with the stirring blade to efficiently supply the medium and remove waste products. 9 after starting culture
The medium was changed on day 4, and then every 4th day. The medium was exchanged by removing all the old medium and adding 450 ml of fresh medium to the flask.

To observe the growth of cells in continuous culture, glucose concentration as nutrients and lactic acid concentration as waste products in the medium were measured every day. Glucose concentration in the medium,
And the lactic acid concentration was measured by a biotech analyzer (AS-210, Asahi Kasei Co., Ltd.) using an enzyme sensor.
In addition, the cell-attached sheet was lightly washed with PBS (Gibco BRL) to increase the ethanol series (70, 80, 90, 95, and 100%).
After dehydrating with, and finally substituting with t-butyl alcohol,
Frozen dryer (ES-2030, Hitachi, Ltd.)
After drying with Pt-Pd by ion sputtering (E-1030, Hitachi Ltd.), SEM (S-4500, Ltd.)
It was observed using Hitachi.

The cell culture bed apatite sheet HAp-SB (175) was incorporated into the spinner flask, and continuous culture of CHO-K1 cells for 21 days was possible. During that time, there was almost no change in pH due to the effect of HEPES buffer. The initial stage of culturing is in the growth phase of cells and the cell density is low, so that the decrease in glucose is small, but it can be seen that the decrease rapidly increases after the first medium exchange (FIG. 15). .
As the glucose concentration decreases due to consumption of glucose, which is a nutrient source, the concentration of lactic acid, a waste product, increases.
The rate of decrease in glucose concentration on the first day after the medium was changed increased from the first time to the third time, which also suggests that cells still tend to increase on the sheet surface. Although the medium was exchanged 3 times and the culture was stopped, it is presumed that the culture can be continued and the cells can be proliferated. It was shown that the cell culture bed apatite sheet is also excellent as a material for continuous culture.

(Example 4) First, a novel cell attachment module using a cell culture bed apatite sheet will be described. Next, a new continuous culture system using the module will be described.

(Observation of CHO-K1 cells on culture bed surface) FIG.
An electron micrograph of CHO-K1 cells grown on the surface of the cell culture bed apatite sheet HAp-SB (85) is shown in FIG. The cells proliferate as if they were spread side by side on the surface of the HAPC forming the sheet. Furthermore, not only the fiber surface of the surface layer of the sheet, but also the fiber surface of a little lower layer is propagated. as a result,
It is considered that 9.1 × 10 ⁶ CHO-K1 cells were grown per sheet. This cell number is about 10 times the cell density in the plastic culture dish (see Table 8). However, cells did not grow in the inner layer of the sheet. This is because the culture this time is static culture in a plastic culture dish.
This is probably because it is limited to the HAPC surface.

Culture Bed HAp-SB (17
5) As a result of observing the cells on the surface with SEM, it was revealed that the cells were not grown on the entire surface of the sheet and there was a part where the cells were not attached yet (FIG. 16).
(B)). It is speculated that the situation was still in the process of growing.
Focusing on the morphology of the cells, the surface is uneven compared to cells in static culture. It appears to form substances that appear to be extracellular matrix on and around the cell surface. In spinner flask culture, since the culture bed sheet is rotated, the medium flows and shear stress is applied to the cells, and it is speculated that the force also acts to peel the cells from the sheet. On the other hand, it is considered that cells may form an extracellular matrix because they are strongly attached to the sheet.

(Proliferation characteristics of CHO-K1 cells) For the purpose of developing an attachment material for animal cells using an apatite-pulp composite, a cell culture bed apatite sheet using the composite as a raw material was prepared. As a result of examining the growth characteristics of CHO-K1 cells using the culture bed as an attachment material, the following matters were clarified.

The cell culture bed apatite sheet has many voids with a size of 10 to 200 μm, which are optimal for cell growth, and is an excellent material as a cell attachment material. The gap is formed by ribbon-shaped HAPC with excellent cell affinity. The cell culture bed apatite sheet is autoclave sterilizable and can be said to be a novel and excellent material for attaching cells.

In the culture using the sheet-shaped culture bed carrier in the culture dish, the cell density on the 7th day of the culture became saturated, and the cell density became 1.5 × 10 ⁶ cells / cm ² . This density is about 10 times higher than the cell density in plastic culture dishes.

CHO-K1 cells could be continuously cultured for 21 days by a novel method in which the sheet-shaped culture bed was incorporated into the stirring blade of a 500 ml pinner flask for large-scale culture and the culture bed itself was rotated. Further continuous culture is considered to be possible. In addition, the cells attached to the surface of the spin-cultured sheet formed a substance considered to be an extracellular matrix, and its morphology was uneven.

(Development of cell culture module using cell culture bed apatite sheet and continuous culture of CHO-K1 cells by bioreactor using the module) The apatite sheet is a sheet-like material and is present on the surface at the same time. Due to the characteristics of apatite, it was expected that it would also have a high affinity for animal cells. Prototype apatite sheet for cell culture bed, and use it as a culture carrier for CH in plastic culture dishes.
As a result of examining the cell growth of O-K1 cells, it was found that the cells grew at a high density on the surface of the carrier.

Therefore, this time, a cell culture bed apatite sheet was used as a material to prepare a culture module usable in a cell culture apparatus capable of continuous culture for a long period of time. A simple cell culture device was prototyped by combining a medium circulation pump, a silicon tube for gas exchange, and the fabricated module, and as a result of continuous culture of CHO-K1 cells, it is possible to culture the cells at high density for a long period of time. Met. Furthermore, as a result of continuous culturing by incorporating this module into a commercially available animal cell continuous culturing device, continuous culturing was continuously possible for about 80 days. The module devised this time has a different design from the conventional culture module, and it is possible to develop it into a module that is more compact and has a higher cell density.

(Preparation of culture module) HAp-SB (85) and H prepared by the above-mentioned cell culture bed apatite sheet preparation method.
Ap-SB (175) was used as the cell culture bed apatite sheet material.

From the results of the above studies, a method of statically culturing CHO-K1 cells by placing a cell culture bed apatite sheet in a plastic culture dish, and incorporating CHO-K1 cells in a spinner flask while rotating the sheet to produce CHO-K1 Both methods of culturing K1 cells were possible. Considering the method of supplying the medium, these methods can be said to be the method in which the medium does not flow or flows in parallel on the sheet surface.
In other words, it is considered that the inflow of the medium into the inside of the sheet is small and the cells do not grow inside.

Considering the cell size, the cell culture bed apatite sheet is capable of adhering / proliferating cells to the internal pores in the thickness direction of the sheet. If cells can be propagated to the inner pores, the effective surface of the cell culture bed apatite sheet will be utilized to the maximum extent, and it will be used more efficiently as a cell attachment material. For that purpose, it is necessary to supply fresh medium to the inside of the sheet,
As a method for this, it is possible to supply the medium so that it will pass through the sheet. Since the cell culture bed apatite sheet is made from HAPC fibers by a papermaking method, unlike the polymer film, there are many pores between the fibers that are connected to each other from the front to the back of the sheet. If the method of circulating the culture medium using these pores is adopted, it becomes possible to supply the fresh culture medium to the surface of the internal pores, and the waste products discharged as a result of cell growth are also efficiently discharged. . Based on the above idea, a cell culture carrier module incorporating a cell culture bed apatite sheet as shown in Fig. 17 (depending on the used cell culture bed apatite sheet, HAp-
We devised HM (85) with SB (85) or HM (175) with HAp-SB (175). this is,
An apatite sheet (diameter) that was cut using a stainless steel syringe holder (KS-25, Advantech Toyo Corp.) in a circle on the support screen.
25mm) and set it for use. Since the main body is made of stainless steel, it can be sterilized by autoclave.

(Cell Culture Device) (Simple Cell Culture Device) A simple cell culture device using a module is shown in FIG. This device is 250ml
(Or 500 ml) glass culture medium bottle, pump for circulating the culture medium, silicon gas exchange tube for supplying oxygen and carbon dioxide gas into the culture medium and assembled with HM (85) or HM (175) module connected. Is. This device is CELL
Based on MAX system (Celleco Inc.). The device is placed in a carbon dioxide incubator and continuously cultured. Oxygen and carbon dioxide in the incubator dissolve into the medium through the gas exchange tube, supplying oxygen to the cells and
The pH is maintained.

(Continuous culture device) In order to establish a long-term culture method rather than simple continuous culture, a HAPC module was installed using a device of a hollow fiber animal cell culture device HFR-1000 (manufactured by Tokyo Rika Co., Ltd.). An attempt was made to continuously culture CHO-K1 cells. The outline of the apparatus is shown in FIG. The configuration of the device is a unit type in which a 1000 ml glass medium bottle, a hollow fiber cell culture cartridge, and a gas exchanger are connected by a Tygon tube and a silicon tube.
Oxygen and carbon dioxide are supplied from a gas exchanger. In addition, a pH sensor, temperature sensor, and DO sensor are incorporated in the flow path of the unit to enable timely measurement of the condition of the medium. This unit is portable and can be autoclaved as it is, and when changing the medium, the medium bottle can be easily replaced by bringing it into the clean bench. At the time of culturing, the unit is placed in a device that is a thermostatic chamber, set in the medium circulation pump incorporated in the device, and the medium is circulated in a hollow fiber cartridge for culturing. This device cultures using a cartridge that uses hollow fibers, but this time, the cartridge was replaced with an HM module to evaluate the performance of the HM module.

(Culture of CHO-K1 cells by cell culture device) (Culture by simplified cell culture device) As the cells (the CHO-K1 cells) used for continuous culture, Ham's F-12 medium containing 10% FBS was used. The cells were precultured at 37 ° C in a carbon dioxide incubator with carbon dioxide concentration 5% / air 95%. A 0.25% trypsin solution (containing 1 mM EDTA) was used as a cell dispersant at the time of cell passage. Cell culture bed apatite sheet HAp-SB (85) (diameter 25 mm, carrier weight 0.042 g) or HAp-SB (175) that was confluent in a 60 mm culture dish several times and set confluent cells in the 60 mm culture dish. ) (Diameter 25 mm, carrier weight 0.086 g). The number of inoculated cells is 3.0 x 1
05 pieces / sheet. The sheet used for culture was previously washed twice with distilled water, sterilized by autoclave, and then washed twice with the medium for continuous culture described below. After inoculation of the cells, the carrier on the 1st day of culture was incorporated into the KS-25 stainless steel cartridge that had been set in the sterilized simple cell culture device. All of these operations were performed in a clean bench. The culture device was placed in a carbon dioxide incubator under the conditions of carbon dioxide concentration 5% / air 95% and culture temperature 37 ° C, and the medium was circulated at 25 ml / min for continuous culture.

The medium used for continuous culture was 10
Ham's F-12 medium containing% FBS (250 ml for HM (85), HM (175)
, 500 ml) and use FBS at certain intervals after the start of culture.
The concentration was reduced to 5% and 2%.

The medium was replaced on the 9th day after the start of the culture, and then the medium was replaced every 4th or 3rd day. The medium bottle was replaced with fresh medium by replacing the medium bottle with old medium with the medium bottle with fresh medium.

(Culturing Method by Continuous Culture Device) According to the simple continuous culture experimental method, the cell culture bed apatite sheet was used.
HAp-SB (85) (diameter 25 mm, carrier weight 0.042 g) was inoculated with 3.0 × 105 CHO-K1 cells and cultured in a carbon dioxide incubator for 1 day. HFR-
It was mounted in a cartridge incorporated in 1000 culture units. The unit was transferred to a culture device, set in a pump, and the medium was circulated at a flow rate of 20 ml / min to start culture. Medium is H
1000 ml of am's F-12 medium (containing 5% FBS) was used. 4 ml of the medium was withdrawn at regular intervals (1 day), and the glucose and lactate concentrations in the medium were measured to confirm the cell growth status.
When the glucose concentration in the medium decreases, fresh medium
The medium was replaced with 1000 ml and continuous culture was continued for 80 days.

(Analysis of Growth Condition) To observe the growth condition of cells in continuous culture, the glucose concentration of nutrients in the medium and the lactic acid concentration of waste products were measured by Biotech Analyzer (AS-210, Asahi Kasei Co., Ltd.). Was measured daily.

After completion of the culture, the cells on the cell culture bed apatite sheet in the module were measured by the following method. First, remove the sheet from the module, wash with 40 ml PBS, and add 1 ml to 7 ml of 0.25% trypsin solution (containing 1 mM EDTA).
After soaking for a minute, thoroughly wash in Ham's F-12 medium containing 10% FBS to remove cells from the sheet. The detached cells were stained with trypan blue staining solution using a hemocytometer and counted.

(Observation of CHO-K1 cells in module) Cell culture bed Adhered and proliferated on the surface or inside the apatite sheet
The morphology of CHO-K1 cells was observed by SEM. PBS sheet
After lightly washing with ethanol, dehydration with ethanol increasing series (70, 80, 90, 95 and 100%), and finally replacing with t-butyl alcohol, use a freeze dryer (ES-2030, Hitachi, Ltd.) After drying, Pt-Pd vapor deposition was performed using ion sputtering (E-1030, Hitachi, Ltd.) and SEM (S-450
0, Hitachi, Ltd. was used for observation.

(Proliferation characteristics of CHO-K1 cells) (Culturing with a simplified cell culture device) A graph showing changes over time in glucose and lactate concentrations in a culture solution continuously cultured with a simplified culture device using HM (175) Shown in 20. In HM (175), glucose decreased only slightly from the initial concentration of 0.18% for the first 3 days after the start of culture, but thereafter decreased steadily, and the consumption decreased from when the concentration fell below 0.08%. . In inverse proportion to that, lactic acid, which is a waste product of cell activity, accumulates. It is considered that growth inhibition occurs when the lactic acid concentration is about 0.07%. It is considered that CHO-K1 cells were proliferating in the inner layer of the sheet during that period. 1, 2
Even after the second medium exchange, the glucose consumption on the first day after the exchange was increased, and it is considered that the cell growth is progressing. After the 4th medium change, the glucose consumption on the first day after the medium change was constant, so the cell density reached a saturated state, and after that, the cells grew while maintaining a certain amount of cells. Presumed to be.

In HAPC (85) M, glucose consumption on the first day after the exchange increased until the third exchange and remained constant thereafter. Since the sheet used for the module is thin, it seems that it took a short time for the cells to reach saturation. After that, the consumption decreased by about 0.59, but it remained constant as HAPC (175) M, indicating that it is growing constantly.

(Culturing Method by Continuous Culturing Device) FIG. 21 shows changes with time of pH, temperature and DO of the medium during continuous culturing.
DO decreases as the culture progresses. When the number of culture days exceeds 40 days, DO becomes 0%, which means that all the oxygen dissolved from the gas exchanger is consumed. Dissolved oxygen seems to be a limiting factor for cell growth.

FIG. 22 shows the daily changes in glucose concentration and lactic acid concentration in the medium during the culture period. For 30 days after the start of culturing, CH is placed in the cell culture bed apatite sheet in the module.
O-K1 cells appear to be proliferating and glucose consumption is increasing. Also, it is in agreement with the tendency of decrease in DO. After 30 days, it seems that the inside of the sheet was also filled with cells, and the consumption became constant. It is considered that the cells were sufficiently grown in the limited void space of the cell culture bed apatite sheet. After this, it is speculated that the constant disappearance and proliferation of cells proceeded. After 50 days, glucose consumption will increase slightly. on the other hand,
In inverse proportion to that, the amount of lactic acid produced decreases. It is thought that this is because the characteristics of the cells have changed slightly. For example, it may have formed a mechanism of resistance to cell growth inhibition by increasing lactate concentration, or
Changes in the lactate biosynthesis pathway may have occurred. However, since we have not investigated what kind of gene has actually been expressed, it cannot be stated at this point, but it is certain that some kind of change in cell characteristics has occurred as a result of long-term continuous culture.

(Comparison of Cumulative Glucose Amount) The glucose consumption amount during the culturing period was converted per unit weight of the culture bed and shown in FIG. 23 as the cumulative consumption amount of the simple continuous culture device and the continuous culture device. In the simple continuous culture device, the cumulative consumptions of the two types of modules having different basis weights were almost the same. The cumulative consumption of the continuous culture device was about twice as large as that of the simple continuous culture device. This is presumably because in the continuous culture device, more oxygen was supplied into the culture solution through the gas exchanger, the number of cells increased, and glucose consumption increased. On the other hand, a culture device using a glass fiber carrier (Perry SD and Wang DIC, Biotechnol. Bioen
g., 34, NO.1, 1 (1989)), the cumulative consumption of continuous culture equipment is 50 times, and that of simplified continuous culture equipment is 22 times. Consumption of glucose, which is a nutrient source, is most closely related to cell density, and the module using the cell culture bed apatite sheet has a higher density than the glass fiber carrier and forms a space for culturing CHO-K1 cells more efficiently. It is believed that

The sheet was taken out from the module after the culturing was stopped, and the number of attached cells was counted and shown in Table 8. The table also shows the maximum number of adherent cells when the same cell culture bed apatite sheet used for continuous culture was placed in a culture dish and CHO-K1 cells were statically cultured on the surface.

[0138]

[Table 8] When used in a module, the number of adherent cells is 5 times or 8 times larger than that of static culture. HM (17
5) was attached about 1.8 times more than HM (85), suggesting the proliferation of cells inside the sheet. On the other hand, in static culture, the effect of sheet thickness on the number of adherent cells was hardly seen, indicating that cells proliferate only on the surface.
An attempt was made to count the cells in HM (85) continuous culture, but it was difficult to distinguish them from the cells because the extracellular matrix was abundant and many fragments were seen in the microscope field. As a result, the number of cells could not be measured. However, considering the size of glucose consumption and the size of the generated cell aggregates, it is assumed that the cell density is more than double that of the culture in the simplified culture device.

(Growth status of CHO-K1 cells in module)
Fig. 24 shows CHO-K1 cells on the surface of the cell culture bed apatite sheet statically cultivated in a plastic culture dish, on the surface of the HM (175) apatite sheet continuously cultivated in a simple type culture device, and on CHO-K1 cells grown therein. The SEM photograph is shown. The CHO-K1 cells on the surface of the statically-cultured sheet are only attached and proliferated on the fiber surface on the surface layer of the sheet, and the proliferation on the fiber surface of the inner layer is not confirmed. In particular, cells proliferate only on the fiber surface, and cells do not propagate in the spaces between the fibers. Also,
The morphology of each cell is clearly visible, and the cell surface is also smooth.

The cells grown on the sheet in HM (175) are attached and grown on the surface and inner layer fiber surfaces. Furthermore, it can be confirmed that the cells have grown to the voids between the fibers. The surface of the cell is uneven with a substance that seems to be extracellular matrix (collagen etc.) attached. The extracellular matrix seems to be a space between the fibers forming the carrier or a support for cells to grow toward the inner layer of the sheet. It is known that cells respond to physical stress in various ways (Bohmann A., Portner
R., Schmieding H., Kasche V. and Markl H., Cytotec
hnology, 9, 51 (1992)). CHO-K1 cells are physically acted upon by medium perfusion, that is, the action of detaching the cells from the cell culture bed apatite sheet is exerted on the CHO-K1 cells, and the cells form a matrix to counteract the cells and strongly attach to the sheet to be detached. It is thought that they tried to prevent Since there is no such stress in static culture, extracellular matrix is not generated, and the cell surface appears smooth in the SEM image.

As a feature of the cells inside the apatite sheet, the shape is more spherical than the cells on the surface. It can be observed that the cells proliferate not only on the HAPC fiber surface but also in the extracellular matrix structure constructed in the fiber voids. In other words, the sheet surface cells are two-dimensionally attached and proliferated in a plane on the fiber surface, but inside the extracellular matrix is constructed, which supports the cells so as to surround them in a three-dimensional manner. It can be seen that cells are proliferating like the tissue of. As a result, CHO- which is an epithelial cell
It is considered that the K1 cells have a spherical morphology inside the sheet. Such three-dimensional cell growth has not been reported in cell culture modules using other fibers, and is a cell growth morphology characteristic of the cell culture bed apatite sheet module. The reason for such proliferation is that the surface of the apatite-pulp composite fiber, which is the surface of the culture bed, is apatite that has excellent adsorption properties for biomolecules such as proteins, and that it has a rough shape that cells are likely to adhere to. It is possible that Furthermore, the voids between HAPC fibers are optimally 10-200 μm for the construction of extracellular matrix.
It is presumed that it is because of the size of.

From these results, the excellent cell culture characteristics of the cell culture bed apatite sheet module were revealed. The characteristics of this module will be discussed in comparison with the fixed bed type cell culture module using the fiber material which has been conventionally studied. Furthermore, based on this stainless steel holder type module, we have created a new module that can continuously culture a large amount of cells for a long period of time.

[0143] Based on the module in which the cell culture bed apatite sheet was incorporated in the stainless steel holder,
The form of the module that can be used for mass culture was prepared as follows.

If an apatite sheet is used as it is, a large-area sheet is required to prepare a large-scale culture bed. For example, 100 g of the cell culture bed apatite sheet HAp-SB (85) has a surface area of 105 × 10 ⁵ cm ² . As a module for incorporating a sheet of this size as it is, it is not practical because it has a large area and a bulky shape. The problem of securing a large area for the medium to pass through in a smaller shape can be solved by folding the sheet and using it as shown in FIG. The sheet is folded into a bellows and placed in a cylindrical holder. By folding, a sheet with a considerable area can be made into a compact shape. Further, by supplying the medium radially from the outside, it becomes possible to allow the medium to pass through the sheet evenly on any surface, which is an advantage of the cell culture bed apatite sheet. This method can be considered as one of the fixed-bed culture devices that have been researched and developed so far. However, in contrast to the fact that these devices are filled with porous cell-attached carriers (microcarriers) for culturing, the apatite sheet module uses folded sheets to generate a concentration gradient in the circulating medium. It has the advantages of being able to control the flow rate and lowering the circulating flow rate of the medium. This sheet folding structure is embodied as a filter cartridge, and it seems that it can be relatively easily realized by diverting its form. If this type of module is realized, it can be expected to become a new cell mass culture apatite sheet module, which enables long-term culture of a large number of cells, and enables efficient production of useful physiologically active substances.

Cell Culture Bed Using the apatite sheet as a material, a novel culture module for animal cell culture was prepared. The module has a structure in which a 25 mm diameter apatite sheet is incorporated in a stainless steel holder, and the medium passes through the inside of the sheet. A simple cell culture device was prototyped by combining a medium circulation pump with a gas exchange silicon tube and module. As a result of continuous culture with a culture device placed in a carbon dioxide incubator, it was possible to continuously culture CHO-K1 cells in the module for 34 to 40 days. The cell density in the module was 1.5 × 10 ⁸ cells / cm ³ per sheet, and it was possible to culture at a high density, and the cell growth was good. In addition, the operability of the culture was simple and efficient, such as the small amount of cells at the start of the culture, and was superior to the existing culture module.

As a result of observing the cells in the module, unlike the stationary culture of the cell culture bed apatite sheet in the culture dish, high density proliferation of cells inside the sheet and formation of extracellular matrix around the cells were observed. The new finding was confirmed. This shows different culture characteristics compared with the culture modules using the conventional fiber materials. Furthermore, by incorporating this module into a commercial cell continuous culture device, continuous culture for 80 days was possible.

(Example 5) A novel module capable of continuously culturing a large amount of cells for a long period was prepared based on a stainless steel holder type module. The new module has a compact shape in which an apatite sheet is folded into a bellows shape and placed in a cylindrical holder. In this module, the medium is evenly supplied to all sides of the sheet, enabling high-density, long-term cell culture. It has become possible to efficiently produce useful physiologically active substances by using a culture device incorporating this module.

Therefore, in this Example, human IL was prepared using a continuous culture device incorporating the apatite module described above.
CHO cells producing (interleukin) -6 were cultured to try human IL-6 production experiment.

Using an HAPC synthesizer, 0.06 mol / L of 15 g of unbeaten commercial softwood bleached kraft pulp (NBKP) was suspended.
HAPC was synthesized by dropping 300 ml of 0.12 mol / L diammonium hydrogen phosphate solution into 1000 ml of calcium nitrate solution. HAPC (sample name is abbreviated as HAPC (NP-0)) was prepared while controlling the pH using a 5 mol / L sodium hydroxide solution at a reaction temperature of 50 ° C. and a reaction pH of 8 as the reaction conditions.

(Preparation of Cell Culture Bed Apatite Sheet and Culture Module) Using HAPC (NP-0) obtained above as a raw material,
The apatite sheet was prepared by drying at 120 ° C using a square sheet machine and a cylindrical electric heater type rotary dryer according to the papermaking method according to the method for producing the apatite sheet. By this operation, most of the unattached apatite particles in the HAPC (NP-0) suspension were removed as a sheet machine filtrate. However, since the apatite particles that were not slightly complexed remained in the sheet, the sheet was disintegrated again with a stirrer to remove it, and this was used as a raw material for the cell culture bed apatite sheet HAPC (NP -0) made into a suspension. Raw material HAPC
(NP-0) 80 WT% and polyolefin type fibrous binder EKC
(Chisso Polypro Fiber Co., Ltd.) 20 WT% was blended, paper was made with a square sheet machine, and then dried in a natural convection dryer without press dehydration. At that time, the drying temperature was set to 135 ° C, which is higher than the melting point of the binder (105 to 130 ° C). Since the rotary dryer used for press-drying conventional apatite sheets is not used, a sheet material with less inter-fiber adhesion and wide voids is created. As a result, a cell culture bed apatite sheet (abbreviated as HAp-SB (85)) with a bulk density of 85 g / m ² and a size of 25 × 25 cm ² having a low bulk density and a high porosity was obtained.

Cell culture bed An apatite sheet HAp-SB (85) was incorporated to prepare a cell culture HAPC module. This uses a stainless steel syringe holder (KS-25, Advantech Toyo Co., Ltd.), and a cell culture bed apatite sheet (diameter 25 mm) cut out in a circle is set on the support screen for use. Since the main body is made of stainless steel, it can be sterilized by autoclave.

(Continuous culture device) The outline of the continuous culture device is shown in FIG. The structure of the continuous culture device is a unit type in which a 1000 ml glass medium bottle, an apatite sheet module, and a gas exchanger are connected by a Tygon tube and a silicon tube. Oxygen and carbon dioxide are supplied from a gas exchanger. In addition, pH may be in the middle of the unit flow path.
A measurement sensor, temperature sensor, and DO sensor are incorporated to enable timely measurement of the condition of the medium. This unit is portable and can be autoclaved as it is, and when changing the medium, the medium bottle can be easily replaced by bringing it into the clean bench. At the time of culturing, put the unit in a device that is a thermostatic chamber,
The medium is circulated in the apatite module by setting it in the medium circulation pump incorporated in the device to carry out culture.

(Cultivation of CHO cells by cell culture device) Human IL-6 is produced on the cell culture bed apatite sheet HAp-SB (85) (diameter 25 mm, carrier weight 0.042 g) according to the above-mentioned simple continuous culture method. CHO cells (Distributed by Professor Mitaka Shirahata, Department of Cellular Control Engineering, Kyushu University)
Abbreviated as 3.0), inoculate 3.0 x 105 cells, and place DM in a carbon dioxide incubator.
After culturing for 1 day using EM medium (containing 5% dialyzed FBS and 50 nM MTX), the cell-attached sheet is placed in a cartridge incorporated in the HFR-1000 culture unit in a clean bench. The unit was transferred to a culture device, set on a pump, and the medium was circulated at a flow rate of 20 ml / min to start culture. Medium is D-MEM medium (containing 5% dialyzed FBS and 50 nM MTX) 1000 ml
Was used. 4 ml of the medium was withdrawn at regular intervals (1 day), and the glucose and lactate concentrations in the medium were measured to confirm the cell growth status. When the glucose concentration in the medium decreased, the medium was replaced with 1000 ml of fresh medium and the continuous culture was continued for 67 days.

(Analysis of Growth Condition) To observe the growth condition of cells in continuous culture, the glucose concentration of nutrients in the medium and the lactic acid concentration of waste products were measured with a Biotech analyzer (AS-210, Asahi Kasei Co., Ltd.). Used daily.

(Analysis of human IL-6) Human IL-6 produced by CHO cells was measured by the ELISA method. A human IL-6 assay kit was used.

(CHO cell growth status) FIG. 26 and FIG.
The changes in glucose concentration and lactic acid concentration in the medium during the culture period are shown. FIG. 26 shows a case (test) in which the DMEM medium was cultured in a low glucose concentration medium at the initial stage of culture and was replaced with a high glucose concentration medium after 40 days and cultured.

At the 47th day, the medium was replaced with a high glucose medium because glucose consumption increased after about 40 days of culturing and the low glucose medium required frequent medium replacement. After the high glucose medium was replaced, glucose consumption became stable, and at that time, it is considered that the cells were sufficiently grown in the sheet.

Based on the result, continuous culture (test) of cells in a high glucose medium was carried out from the initial stage of culture.

FIG. 27 shows the case where the DMEM medium was cultured in the high glucose concentration medium from the initial stage of the culture.
It was found that glucose consumption was high from the early stage of culture and lactic acid production was high. But 4
From day 0, the consumption was almost the same as in the test in which the low glucose medium was replaced with the high glucose medium. It was inferred that the cell mass did not differ so much.

(Production of Human IL-6) FIGS. 28 and 29 show changes in human IL-6 concentration in the culture medium.

In the test, the human IL-6 concentration after changing to the high glucose medium changed highly. It is inferred that immediately after changing to a high glucose medium, the cell characteristics changed to adapt to the environment, and the productivity of human IL-6 per cell increased.

In the test, no aspect of abrupt changes in human IL-6 concentration is observed. It is considered that the concentration of human IL-6 changes depending on the amount of cells in the module. Figure 30
Shows the cumulative production of human IL-6. In the test, the cumulative production of human IL-6 was 73000 μg at the end of culture, and in the test it was 62000 μg. The difference is based on the difference in the production amount after changing from the low glucose medium to the high glucose medium. Assuming that the number of cells is the same, it is considered that the productivity of one cell was enhanced.

[0163]

EFFECTS OF THE INVENTION Apatite-pulp composite fiber (HAPC), which is a novel fiber material having a structure in which HAp is crystallized on the pulp surface using the wet synthesis reaction of HAp and the pulp surface is coated with HAp Was developed. HAPC has pulp core and HAp
It has a double structure of sheath and can be said to be an inorganic-organic composite material. If we focus only on the HA p on the surface, it is a HA p fiber material. Up until now, HAp had been developed as a ceramic fiber, but there were problems in handling such as short fiber length and narrow width. HAPC has the same morphology as wood pulp, so it has excellent handleability and enables the use of HAp fibers in various applications.

HAPC also means functionalization of pulp with an inorganic material. The pulp functionalization method using HAp is a unique new method, compared with the attempts to make functional pulp by combining inorganic powder with pulp, which has been done so far. The method of making HAPC is an insoluble inorganic salt
Let HA p crystallize on the pulp surface. For this reason, HAPC can be easily compounded with commercially available dry pulp, unlike the conventional pulps that have no drying history used for the production of inorganic powder-composite functional pulp. Since the actual compounding method is relatively simple, it is a technology that even small and medium-sized Japanese paper companies can introduce.

An apatite sheet and a method for manufacturing the same have been developed. The apatite sheet was made into a sheet using a papermaking method, which is a method of producing paper using HAPC as a raw material fiber.
This method is simpler in operation and lower in cost than conventional methods for producing inorganic powder paper. Since HAPC is synthesized and used as the raw material for the sheet, HAp is synthesized and blended into the pulp at the same time, which simplifies the operating procedure. Furthermore, HAp can be fixed to pulp and formed into a sheet without using a polymeric fixing agent, which was required in the conventional method. This technology can also be introduced to mechanical Japanese paper companies in Fukuoka Prefecture, and companies in the prefecture actually succeeded in producing apatite sheets using their own papermaking equipment.

[0166] The apatite sheet is a sheet material provided with HAp ion adsorption characteristics and biocompatibility characteristics. HAp, which was previously used only for inorganic powders, can now be used as a sheet material. It was revealed that the apatite sheet is excellent as a material that adsorbs biological components such as proteins and nucleic acids. In the future, it is expected to be used for chemical analysis sheets, etc. that take advantage of these characteristics. In addition to such special applications, it can be applied to adsorbent materials for masks and adsorptive function shoji paper, and is expected to be a great help to new product development for mechanical Japanese paper companies.

The cell culture bed apatite sheet was developed by applying the manufacturing technology of the apatite sheet. This sheet is HA
It is a new cell-adhesive material that takes advantage of both the characteristics of PC fibers and the characteristics of laminated sheet materials. HAPC
Its surface is HAp, so it has excellent cell affinity. Since it is a sheet in which HAPC fibers in the form of pulp fibers are laminated, the optimal size for cell attachment (10 to 200 μm)
In addition, it has a large area of inter-fiber voids. Since it has a sheet shape, it can be molded into any shape, so it can be used for static culture in a culture dish, and can also be used for long-term rotary culture by incorporating it into a stirring blade in a spinner flask. Spinning culture in a spinner flask is the world's first culture method, and rotating the sheet enables efficient supply of nutrients and removal of waste products.

Cell Culture Bed A cell culture module utilizing an apatite sheet was developed for the first time. In this module, the cell culture bed apatite sheet incorporated is supplied so that the medium can pass through, and the cells grow to the inside of the sheet. This utilization method takes advantage of the characteristic of continuous inter-fiber voids that an apatite sheet has. We have developed a continuous cell culture device incorporating a module. It can be said that this device is a kind of fixed bed culture system. Using this device, we succeeded in long-term high-density continuous culture of CHO-K1 cells. It was possible to continuously culture CHO-K1 cells in the module for 40 days up to 80 days. The cell density in the module was 1.5 × 10 ⁸ cells / cm ³ per sheet, and it was possible to culture at a high density, and the cell growth was good. Characteristically, the cells inside the sheet produced extracellular matrix and proliferated three-dimensionally. Moreover, the operability of the culture was simple and efficient, such that the amount of cells at the start of culture was small. For the first time in the world, we proposed a new design of a module for continuous mass culture that is an extension of the idea of the prototype module. The feature of this module is that the sheet is folded into a bellows shape and incorporated into a cylindrical holder. By folding, a sheet with a considerable area can be made into a compact shape. Furthermore, by supplying the medium radially from the outside, it is possible to allow the medium to pass through the sheet evenly on any surface. This module enables highly efficient production of useful physiologically active substances such as interferon using animal cells, and has great promise as a new modeling device for new artificial organs using cultured cells.

In the previous research on the apatite sheet cell culture bed module, it was revealed that CHO-K1 cells were continuously cultured at high density for a long period of time. Furthermore, it was also found that human IL-6-producing CHO cells can be continuously cultured using the module, and that useful bioactive substances including cytokines such as human IL-6 can be efficiently produced.

Regarding the state of the cells in the module, it seems that the cells proliferate and grow up to the inner layer of the apatite sheet, the cells are three-dimensionally cultured, and the cells look like a cell tissue. This is rarely observed in other culture methods, and can be said to be an excellent feature of the cell culture bed apatite sheet module. If this characteristic is utilized, it can be used not only as a cell culture module for protein production, but also as an artificial biological tissue replacement module in which cells of various species that can be used when analyzing the effect of pharmaceuticals on cell tissues are analyzed. To be

[Brief description of drawings]

FIG. 1 is a schematic diagram of a HAPC synthesizer.

FIG. 2 shows X-ray patterns of pulp and HAPC.

FIG. 3 shows HAPC (N- (8,9,10)-(1-
5)) X of HAp and cellulose due to difference in synthesis conditions
The graph which shows a line intensity ratio.

FIG. 4 shows HAPC (N- (8,9,10)-(1-
5) A graph showing changes in the amount of HAp attached due to differences in the synthesis conditions.

FIG. 5: HAPC (N-8-1), HAPC (N-8-
4) and an SEM photograph showing the surface structure of NBKP.

FIG. 6 is a graph comparing the HAp adhesion amount of the composite synthesized by the HAPC method and the HAp adhesion amount of the apatite-fixed pulp prepared by the strong stirring method.

FIG. 7 is a diagram showing a comparison between the HAPC method and a conventional method for producing an inorganic powder sheet.

FIG. 8 is an SEM photograph of an apatite sheet (NP-1, LP-1).

FIG. 9 is a graph showing X-ray diffraction patterns of a pulp sheet and an apatite sheet.

FIG. 10 is a graph showing the protein and nucleic acid adsorption properties of the apatite sheet.

FIG. 11 is a schematic view showing a spinner flask in which an apatite sheet for a cell culture bed is wrapped around a stirring blade.

FIG. 12 is a graph showing a comparison of pore volume distributions of a cell culture bed apatite sheet and an apatite sheet.

FIG. 13 is an SEM photograph of a cell culture bed apatite sheet.

FIG. 14 is a graph showing changes in cell density of expanded CHO-K1 cells.

FIG. 15 is a graph showing changes in glucose and lactate concentrations in continuous cell culture using a spinner flask.

FIG. 16 is an SEM photograph showing CHO-K1 cells grown on the surface of the cell culture bed apatite sheet. (A) Static culture in a culture dish (B) Spinning culture in a spinner flask.

FIG. 17 is an exploded view showing a cell culture carrier module incorporating a cell culture bed apatite sheet.

FIG. 18 is a view showing a simple cell culture device using the module of FIG.

19 is a schematic view showing a bioreactor for continuous culture using the module of FIG.

FIG. 20 is a graph showing changes over time in glucose consumption and lactic acid production in a simple cell culture device.

FIG. 21 is a graph showing changes in pH, temperature and DO during continuous culture.

FIG. 22: CH in continuous culture device using module
The graph which shows the time-dependent changes of the glucose consumption amount and lactic acid production amount of O-K1 cells.

FIG. 23 is a graph showing cumulative glucose consumption in a simple cell culture device and a continuous culture device.

FIG. 24 shows the morphology of CHO-K1 cells in the cell culture bed apatite sheet surface in a simple cell culture device and the C of the cell culture bed apatite sheet surface in static culture.
The SEM photograph which shows the form of HO-K1 cell.

FIG. 25 is a view showing a module for high-density continuous culture, which incorporates a cell culture bed apatite sheet.

FIG. 26 is a graph showing daily changes in glucose concentration and lactic acid concentration in the medium during the continuous culture period.

FIG. 27 is a graph showing changes over time in glucose concentration and lactic acid concentration in the medium during the culture period.

FIG. 28 is a graph showing the human IL-6 concentration in the culture medium when a low glucose medium and a high glucose medium were used.

FIG. 29 is a graph showing the human IL-6 concentration in the culture medium when a high glucose medium was used.

FIG. 30 is a graph showing the cumulative production of human IL-6 when a low glucose medium and a high glucose medium were used.

Continued front page    (72) Inventor Hironobu Kawakatsu             405-3, Vol. 1, Okawa, Fukuoka Prefecture Industrial Technology, Fukuoka Prefecture             Surgery Center Interior Research Institute (72) Inventor Hiroshi Kanegae             1465-5 Aikawacho, Kurume City, Fukuoka Prefecture             Industrial Technology Center Biofood Research Institute (72) Inventor Minoru Shirahata             1-21-9 Maisato, Koga City, Fukuoka Prefecture F term (reference) 4B029 AA02 AA21 BB11 CC10 DA10                 4B065 AA90X BC22 BC41

Claims (7)

[Claims] 1. A method for high-density cell culture of cells using an apatite sheet, which comprises culturing cells at a high density using a culture device incorporating an apatite sheet. 2. The high-density cell culture method for cells according to claim 1, wherein the cells are continuously cultured, and a high-density cell culture method for cells using an apatite sheet. 3. The method for high-density cell culture of cells according to claim 1 or 2, wherein the cells are animal cells, and a high-density cell culture method for cells using an apatite sheet. 4. A method for high-density cell culture of cells according to any one of claims 1 to 3, wherein an apatite sheet is used which is a cell that produces physiological activity. High-density cell culture method for cells. 5. A high-density cell culture device in which a cell culture module incorporating an apatite sheet is arranged. 6. The high-density cell culture device according to claim 5, wherein the apatite sheet is incorporated in a folded shape so as to increase its surface area. 7. A cell culture module which is a cell culture module incorporating an apatite sheet.