CN108102915A - A kind of mediate contact co-culture system for being engineered amplification - Google Patents

Abstract

The invention discloses an engineerable and scalable indirect contact co-cultivation system and a preparation method thereof. The system is composed of a bioreactor containing many tiny unit particles. The tiny unit particles are spherical hydrogel carriers with a particle size of 100-1000 microns, and the surface of the hydrogel ball has a layer of hydrogel film structure. One type of cells is inside the spherical hydrogel, and the other type of cells is distributed on the surface of the spherical hydrogel membrane, forming two types of cells that can contact the cell membrane through the membrane pores of the hydrogel membrane, and facilitate the indirect contact of downstream separated cells. culture unit.

Description

An indirect contact co-culture system that can be engineered and scaled up

technical field

The invention relates to an indirect contact co-cultivation system, in particular to an indirect contact co-cultivation system characterized by a spherical membrane.

Background technique

Cell co-culture technology provides the possibility for the in vitro study of cell biology. According to the degree of contact between co-cultured cells, the current co-culture technology is divided into three types: direct contact co-culture, non-contact co-culture and indirect contact co-culture. Among them, the direct contact co-culture technology is beneficial to the function of the cells due to the full contact between the co-cultured cells, but the disadvantage is that after the co-cultivation is over, it is difficult to separate the two cells, and only one of the cells can be fluorescently labeled. Separation by flow sorting, or by magnetic field separation after one of the cells has been labeled with immunomagnetic beads, is tedious and expensive. In contrast, the non-contact co-culture technique is relatively easy to separate the two types of cells, but the disadvantage is that the stromal cells do not show significant support functions for the parenchymal cells due to the absence of contact between the two types of cells. The direct contact and non-contact advantages of the indirect contact co-culture technology show advantages, which are conducive to both the support function of stromal cells and the separation of co-cultured cells. At present, there are two types of cell indirect contact co-culture technologies that have been disclosed: one is to inoculate two kinds of cells on the upper and lower surfaces of the membrane through the transwell flat membrane to construct an indirect contact co-culture system, which is regulated by regulating the porosity of the membrane. The degree of contact between cells. The other is to process a pair of comb-like structures that can be inserted into each other through micro-processing technology, and inoculate two types of cells co-cultured on the surface of each comb-like structure to construct an indirect contact co-culture system. The depth of the comb-like structure regulates the degree of contact between cells, and the cultured cells are separated by separating the pair of comb-like structures. The biggest problem with these two culture methods is that their indirect contact co-culture system can only be used for laboratory research, it is difficult to achieve engineering scale-up, and cannot meet the production needs of cell products.

Contents of the invention

The technical scheme adopted by the present invention to solve the above-mentioned technical problems is: to provide an indirect contact co-cultivation system that can be engineered and scaled up. A spherical hydrogel carrier with a diameter of 100-1000 microns, the hydrogel carrier is a shell-core structure with a layer of hydrogel film on the surface, one type of cells is inside the spherical hydrogel, and the other type of cells is distributed in the spherical water On the surface of the gel membrane, cell contact between the two types of cells can occur through the pores of the hydrogel membrane, and it is convenient for the cells separated downstream of the two types of cells to indirectly contact the co-culture unit.

Further, the bioreactor is a fluidized bed bioreactor, a fixed bed bioreactor, a stirred bioreactor or a rotary bioreactor.

Further, the total volume of the tiny unit particles distributed in the bioreactor occupies 10-90% of the volume of the bioreactor.

Further, the spherical hydrogel carrier includes alginate, chitosan, and one or more of collagen, matrigel, chondroitin sulfate, and fibronectin.

Further, the shell part of the spherical hydrogel carrier is a polyelectrolyte complexed hydrogel film formed by electrostatic crosslinking between chitosan and alginate mixture, the film thickness is below 30 microns, and the film pores are Below 5 microns.

Further, in the spherical hydrogel carrier, the alginate includes one or both of calcium alginate or barium alginate.

The method for preparing tiny unit particles is as follows:

A, prepare the alginate solution (one or both in sodium alginate or potassium alginate) of concentration 10-50g/L;

B. Collagen solution, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution with a concentration of 2-50g/L are prepared;

C. Preparation of gel bath aqueous solution: the gel bath contains one or two kinds of calcium chloride or barium chloride with a total concentration of 5-30g/L; it also contains sodium chloride or potassium chloride with a total concentration of 1-20g/L One or two of them; it also contains one or more of the total concentration of 0.5-20g/L sodium citrate or sodium phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate; it also contains a total concentration of 0-30g/L One or two of L Tween or F68;

D, preparation concentration 1-10g/L chitosan solution;

E, uniformly mix the sodium alginate solution prepared in step A with one or more of the collagen solution prepared in step B, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution, the mixed solution The concentration of sodium alginate in the medium is kept above 10g/L;

F. Evenly disperse the collected cell a into the mixed solution prepared in step E, pass the mixed solution containing cell a through fluid granulation technology to form uniform droplets, and the droplets enter the gel bath solution prepared in step C, and at the same time Combining with temperature changes (if it contains collagen components, increase the temperature to above 37°C), a gelation reaction occurs, that is, the core part of the spherical hydrogel carrier embedded with cells a is obtained;

G. React the core part of the spherical hydrogel carrier embedded with cell a obtained in step F with the chitosan solution prepared in step D to form a film, and the volume ratio of the carrier to the chitosan solution is 1:2-1:10 , let the carrier be in a uniform suspension state in the chitosan solution, and carry out the polyelectrolyte complexation reaction for 2-60 minutes, that is, a hydrogel film is formed on the surface of the spherical hydrogel carrier inner core, and the shell core is obtained after washing with normal saline or PBS solution Structured cells a distributed inside the hydrogel carrier;

H. Co-incubate cell b with the hydrogel carrier embedded with cell a prepared in step G for 1-24 hours to obtain one type of cell inside the spherical hydrogel and another type of cell distributed on the surface of the spherical hydrogel membrane Indirect contact co-cultivation of tiny unit particles.

Further, in the preparation process of the spherical hydrogel carrier, the fluid granulation technology is high-voltage electrostatic field technology, gas orifice spray technology, microfluidic technology or emulsification technology, and the prepared spherical hydrogel carrier particles monodisperse.

The indirect contact co-cultivation system that can be engineered and enlarged can be used for in vitro indirect contact co-cultivation of hepatocytes and stromal cells; in vitro indirect contact co-cultivation of stem cells and induced cells; in vitro indirect contact co-cultivation of stem cells and trophoblast cells; Indirect contact co-culture of tumor cells and stromal cells in vitro and other systems that require co-cultivation of two kinds of cells, and after co-cultivation, trypsin can digest the cells distributed on the surface of micro-unit particles to realize the separation of co-cultured cells .

Beneficial effects of the present invention:

1. The indirect contact co-culture system, through the construction of a spherical membrane, greatly increases the membrane area per unit volume of the carrier, and the spherical particle size is 100-1000 microns, which is more conducive to engineering than the disclosed flat membrane (transwell membrane) technology enlarge;

2. Cell a grows in a three-dimensional state inside the spherical hydrogel, which more simulates the microenvironment in vivo and is conducive to the physiological function of cells;

3. Micro unit particles composed of spherical hydrogel microspheres can be cultivated in various bioreactors to achieve scale-up;

4. After the co-cultivation, the cells b distributed on the surface of the spherical hydrogel carrier can be digested by trypsin, so as to realize the separation of cells a and b, which can be studied separately or used as cell products.

Description of drawings

Fig. 1 is a schematic diagram of the preparation method of tiny unit particles in an indirect contact co-culture system.

FIG. 2 is a schematic diagram of the experimental design of the experimental group and the two control groups in Example 1: the schematic diagram is a cross-section of a bioreactor loaded with tiny unit particles.

Among them, the micro-unit particles in Figure A are indirect contact co-culture micro-unit particles; the micro-unit particles in Figure B are the control group 1, that is, the micro-unit particles loaded with liver cells alone; A non-contact co-culture system of micro-unit particles (small particle size) loaded with hepatocytes and micro-unit particles (large particle size) loaded with vascular endothelial cells alone.

Detailed ways

Fluid granulation techniques include: electrostatic droplet method ^[1] , orifice extrusion method ^[2] , emulsification-external gelation method ^[3] , emulsification-internal gelation method ^[4] , membrane emulsification method ^{[5] ]} , microfluidic method ^[6] .

Example 1

【1】Prepare sodium alginate solution with a concentration of 30g/L;

[2] Prepare a collagen solution with a concentration of 5g/L and adjust it to neutral with NaOH;

[3] Prepare the gel bath solution, which contains calcium chloride (concentration 7g/L), sodium chloride (concentration 3g/L), sodium dihydrogen phosphate (concentration 1g/L) and F68 solution (1g/L) in the gel bath. L);

【4】Preparation of chitosan solution, the concentration is 5g/L;

[5] Mix the sodium alginate solution prepared in step [1] with the collagen solution prepared in step [2] in a volume ratio of 2:1;

[6] Collect the hepatic parenchymal cells cultured to the logarithmic growth phase by centrifugation, and evenly disperse them into the mixed solution prepared in step [5] at a density of 1×106/ml;

[7] The mixture of sodium alginate and collagen containing hepatic parenchymal cells prepared in step [6] forms a stable jet under a high-voltage electrostatic field, sprays it into the gel bath prepared in step [3], and The gel bath was implanted into the cell incubator, and after gelation reaction at 37°C for 30 minutes, the sodium alginate-collagen blended hydrogel microspheres embedded with hepatic parenchymal cells were prepared;

[8] React the microspheres prepared in step [7] with chitosan solution for 10 minutes, wash with saline 3 times, and obtain the alginate-collagen-shell with a putamen structure embedded with hepatic parenchymal cells Polysaccharide microspheres, the particle size of the microspheres is 300±100 microns;

[9] Digest the vascular endothelial cells cultured to the logarithmic growth phase, and mix the alginate-collagen-chitosan microspheres containing the putamen structure of hepatic parenchymal cells prepared in step [8] according to 1×105 cells Ratio of /ml microspheres, co-cultivate in the culture dish pre-treated with agarose, co-incubate in the cell culture box, shake regularly to make the vascular endothelial cells evenly adhere to the surface of the microspheres. The schematic diagram of the whole preparation process is shown in Figure 1.

Control group 1 is the alginate-collagen-chitosan microspheres prepared in step [8] embedded with the putamen structure of hepatic parenchymal cells (hepatocyte culture group alone);

Control group 2 is to prepare vascular endothelial cells in alginate-collagen-chitosan microspheres with a particle size of 800±100 microns according to steps [1]-[8] (non-contact co-culture group). The schematic diagram of the experimental design of the control group is shown in Figure 2.

[10] Mix the alginate-collagen-chitosan hydrogel microspheres prepared in step [9] with hepatic parenchymal cells inside and vascular endothelial cells on the surface with the medium at a volume ratio of 1:5 and stir cultured in a bioreactor.

In control group 1, the alginate-collagen-chitosan hydrogel microspheres that only embed hepatic parenchymal cells were mixed with the medium at a volume ratio of 1:5 and cultured in a stirred bioreactor.

For control group 2, the 800-micron particle size microspheres embedded with vascular endothelial cells and the 300-micron particle size microspheres embedded with hepatic parenchymal cells prepared in step [8] were blended according to the volume ratio of 1:10, and the microspheres were mixed. The total volume of the spheres was mixed with the medium in a volume ratio of 1:5 and cultured in a stirred bioreactor.

【11】After the culture is over, the hydrogel microspheres can be collected by natural sedimentation. After washing, the vascular endothelial cells adhered to the surface of the microspheres are digested with trypsin, and the hydrogel containing cultured and expanded hepatic parenchymal cells is left Microspheres for the study of in vitro bioartificial liver support systems.

For control group 1, the hydrogel microspheres embedded with hepatic parenchymal cells can be collected directly by natural sedimentation.

In control group 2, hydrogel microspheres with two particle sizes collected by natural sedimentation were physically separated through a sieve with a pore size of 600 microns to obtain hydrogel microspheres with a particle size of 300 microns and only embedded hepatic parenchymal cells.

[12] Take a small amount of indirect contact co-cultured microspheres after step [11], and use the method of reference ^[7][8][9] to detect the function of hepatic parenchymal cells. The results show that the hepatocytes obtained by indirect contact co-culture Compared with the hepatocytes obtained from the single culture group and the non-contact co-culture group, albumin secretion and urea synthesis ability were significantly enhanced in the indirect contact co-culture group; The expression levels of apolipoprotein-related gene (ApoA-1), hepatocyte nuclear transcription factor (HNF-4α) and albumin gene were significantly enhanced in the indirect contact co-culture group; group was significantly enhanced.

Example 2

【1】Prepare sodium alginate solution with a concentration of 40g/L;

[2] Prepare a collagen solution with a concentration of 4g/L and adjust it to neutral with NaOH;

[3] Prepare the gel bath solution, which contains calcium chloride (concentration 7g/L), sodium chloride (concentration 3g/L), sodium dihydrogen phosphate (concentration 1g/L) in the gel bath;

【4】Preparation of chitosan solution, the concentration is 5g/L;

[5] Mix the sodium alginate solution prepared in step [1] with the collagen solution prepared in step [2] in a volume ratio of 1:2;

[6] Centrifuge the fetal rat fibroblasts cultured to the logarithmic growth phase, and uniformly disperse them into the mixture prepared in step [5] at a density of 2×10 ⁵ /ml;

[7] The mixture of sodium alginate and collagen containing fetal mouse fibroblasts prepared in step [6] and the gel bath solution prepared in step [3] were passed through the microfluidic T-shaped channel to prepare the particle size For 300-micron pre-gel particles, collect the pre-gel particles in the gel bath prepared in step [3], put them in the cell culture incubator, and gelatinize at 37°C for 30 minutes to prepare the embryo-embedded cells. Sodium alginate-collagen blended hydrogel microspheres of mouse fibroblasts;

[8] The microspheres prepared in step [7] were reacted with chitosan solution for 10 minutes, washed with normal saline for 3 times, and then the alginate-collagen with a putamen structure embedded with fetal mouse fibroblasts was obtained. -Chitosan microspheres, the particle size of the microspheres is 300±30 microns; after 5 days of culture, the cells enter the end of the logarithmic growth phase, and the gel microspheres are collected;

[9] Human embryonic stem cells, and the alginate-collagen-chitosan microspheres containing the putamen structure of fetal mouse fibroblasts prepared in step [8] according to the ratio of 2×10 ⁵ cells/ml microspheres , co-cultivate in the pre-treated culture container with agarose, co-incubate in the cell culture box, shake regularly to make the stem cells evenly adhere to the surface of the microsphere;

[10] Mix the alginate-collagen-chitosan hydrogel microspheres prepared in step [9] with fetal mouse fibroblasts inside and human embryonic stem cells adhered to the surface with the medium at a volume ratio of 1:5 Cultivated in a fluidized bed bioreactor;

[11] After the culture is over, the hydrogel microspheres can be collected by natural sedimentation, and the human embryonic stem cells amplified on the surface of the microspheres by trypsin after washing are detected to detect the expression of stemness-related genes, and it is found that Sox2/Nanog/Oct4 There is no difference from the P1 generation.

references:

【1】In Vivo Culture of Encapsulated Endostatin-Secreting Chinese Hamster Ovary Cells for Systemic Tumor Inhibition.Human Gene Therapy.2007,18:474-481;

[2] A preparation method of high-economic fish microsphere opening bait, Chinese invention patent, 200510136769.7;

【3】Preparation of lactic acid bacteria-enclosing alginate beads inemulsio-n system: effect of preparation parameters on bead characteristics, Polym.Bull, 2009, 63: 599-607;

【4】Preparation of immobilized yeast microcapsules by emulsification-internal gelation process, Chinese Journal of Chemical Industry, 2009, 60(3): 710-717;

【5】Preparation of uniform calcium alginate gel beads by membrane emu-lsification coupled with internal gelation. Journal of Applied Polymer Science, 2003, 87(5): 848-852;

【6】Microfluidic Generation of Monodisperse, Structurally Homogeneous Alginate Microgels for Cell Encapsulation and 3D Cell Culture, Adv.HealthcareMater.2015,4(11):1628-1633;

【7】Fabrication of stable galactosylated alginate microcapsules viacovalent coupling onto hydroxyl groups for hepatocytes applications. Carbohydrate Polymers, 2017, 456-465;

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Claims (9)

1. An indirect contact co-cultivation system that can be engineered and enlarged is characterized in that the system is composed of a bioreactor equipped with many tiny unit particles, and its tiny unit particles are spherical hydrogels with a particle size of 100-1000 microns Glue carrier, the hydrogel carrier has a shell-core structure with a layer of hydrogel film on the surface, one type of cells is inside the spherical hydrogel, and the other type of cells is distributed on the surface of the spherical hydrogel film, forming a gap between two types of cells. Cell contact can occur through the pores of the hydrogel membrane, and it is convenient for the cells separated downstream of the two cells to indirectly contact the co-culture unit. 2. A kind of indirect contact co-cultivation system that can be enlarged by engineering as claimed in claim 1, is characterized in that, described bioreactor is fluidized bed bioreactor, fixed bed bioreactor, stirring type bioreactor bioreactor or rotary bioreactor. 3. A kind of indirect contact co-cultivation system that can be enlarged by engineering as claimed in claim 1, is characterized in that, the total volume of the tiny unit particles distributed in the bioreactor occupies 10-90% of the volume of the bioreactor. %. 4. A kind of indirect contact co-culture system that can be enlarged by engineering as claimed in claim 1, is characterized in that, the composition of described spherical hydrogel carrier comprises alginate and chitosan; Also contains collagen, One or more of matrigel, chondroitin sulfate, and fibronectin. 5. A kind of indirect contact co-cultivation system that can be engineered enlarged as claimed in claim 4, is characterized in that, the shell part of described spherical hydrogel carrier is formed by static electricity between chitosan and alginate mixture. The polyelectrolyte complex hydrogel membrane formed by cross-linking has a film thickness of less than 30 microns and a membrane pore of less than 5 microns. 6. A kind of indirect contact co-cultivation system that can be engineered and enlarged as claimed in claim 4 is characterized in that, in the spherical hydrogel carrier, the alginate comprises calcium alginate or barium alginate one or two of. 7. A kind of engineerable amplified indirect contact co-cultivation system as claimed in claim 1, characterized in that, the preparation method of said tiny unit particles comprises the following steps: A, prepare the alginate solution of concentration 10-50g/L, described alginate is one or both in sodium alginate or potassium alginate; B. Collagen solution, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution with a concentration of 2-50g/L are prepared; C. Preparation of gel bath aqueous solution: the gel bath contains one or two kinds of calcium chloride or barium chloride with a total concentration of 5-30g/L; it also contains sodium chloride or potassium chloride with a total concentration of 1-20g/L One or two of them; it also contains one or more of the total concentration of 0.5-20g/L sodium citrate or sodium phosphate or disodium hydrogen phosphate or sodium dihydrogen phosphate; it also contains a total concentration of 0-30g/L One or two of L Tween or F68; D, preparation concentration 1-10g/L chitosan solution; E, uniformly mix the alginate solution prepared in step A with one or more of the collagen solution prepared in step B, chitosan solution, matrigel solution, chondroitin sulfate solution or fibronectin solution, the mixed solution The concentration of alginate in the medium is kept above 10g/L; F. Evenly disperse the collected cell a into the mixed solution prepared in step E, pass the mixed solution containing cell a through fluid granulation technology to form uniform droplets, and the droplets enter the gel bath solution prepared in step C, and at the same time Combined with the temperature change, a gelation reaction occurs, and the core part of the spherical hydrogel carrier embedded with cell a is obtained; G. React the core part of the spherical hydrogel carrier embedded with cell a obtained in step F with the chitosan solution prepared in step D to form a film, and the volume ratio of the carrier to the chitosan solution is 1:2-1:10 , let the carrier be in a uniform suspension state in the chitosan solution, and carry out the polyelectrolyte complexation reaction for 2-60 minutes, that is, a hydrogel film is formed on the surface of the spherical hydrogel carrier inner core, and the shell core is obtained after washing with normal saline or PBS solution Structured cells a distributed inside the hydrogel carrier; H. Co-incubate cell b with the hydrogel carrier embedded with cell a prepared in step G for 1-24 hours to obtain one type of cell inside the spherical hydrogel and another type of cell distributed on the surface of the spherical hydrogel membrane Indirect contact co-cultivation of tiny unit particles. 8. A kind of indirect contact co-cultivation system that can be engineered and enlarged as claimed in claim 7 is characterized in that, in the preparation process of the spherical hydrogel carrier, the fluid granulation technology is high-voltage electrostatic field technology, gas sharp Hole spray technology, microfluidic technology or emulsification technology, the particle size of the prepared spherical hydrogel carrier is monodisperse. 9. According to claim 1, the indirect contact co-culture system that can be engineered and enlarged is characterized in that, the described amplified engineered indirect contact co-culture system can be used for indirect contact co-culture of hepatocytes and stromal cells in vitro. Culture; in vitro indirect contact co-culture of stem cells and induced cells; in vitro indirect contact co-culture of stem cells and trophoblast cells; in vitro indirect contact co-culture of tumor cells and stromal cells, and after co-cultivation, trypsin can be distributed in micro-unit particles The cells on the surface are digested to achieve the separation of co-cultured cells.