CN103469309B - A kind of tissue homogenate method is separated the method that viable cell builds cell bank - Google Patents
A kind of tissue homogenate method is separated the method that viable cell builds cell bank Download PDFInfo
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Abstract
The invention discloses a kind of tissue homogenate method isolating fetal appurtenant histocyte and build the method for cell bank, when object is to build cell bank step simple, save time, be easy to Quality Control and standardization.The present invention is made up of following step: the cleaning of (1) fetal appendage tissue; (2) the homogenate pre-treatment of tissue; (3) tissue homogenate process isolated cell (group); (4) collection of the filtration treatment organized after homogenate and cell (group); (5) cell (group) detects; (6) cell (group) of frozen separation, builds cell bank.Compared with the conventional method, the inventive method have simple to operate, the used time is short, do not introduce exogenous agent, be easy to the advantages such as stdn and quality control and cell acquisition amount are large, for improving the structure efficiency of fetal appendage cell bank, the stem cell that acquisition high quality is used for the treatment of has great importance.
Description
Technical Field
The invention relates to a method for constructing a cell bank of fetal accessory tissue, in particular to a physical method for constructing a cell (cluster) bank by separating cells from fetal accessory tissue.
Background
The fetal appendage tissue mainly comprises placenta lobules, placenta base, decidua basalis, amnion and umbilical cord tissue. The placenta, which originates from the extraembryonic mesoderm during the embryonic development, is composed of mesenchyme, blood vessels and trophoblasts, is the earliest hematopoietic organ of the fetus, and contains a large amount of mesenchyme components. The latest research shows that the placenta contains mesenchymal stem cells, placenta sub-totipotent stem cells and abundant hematopoietic stem cells, and the separation and culture of the pluripotent stem cells from the placenta opens up a brand-new and rich source for experimental research and clinical application.
The placental sub-totipotent stem cells are derived from placental tissues, appear from the 5 th to 7 th days of embryogenesis, can be differentiated to form more than 200 human tissue organ cells, but cannot form a complete human body. The pluripotent stem cells have many biological characteristics, and can be directionally differentiated into fat cells, osteoblasts, nerve cells, liver epithelial cells and the like in vitro.
Placenta hematopoietic stem and fine powderThe cells areA group of primitive hematopoietic cells present in placental tissue is the progenitor of blood cells (erythrocytes, leukocytes, platelets, etc.), is a highly undifferentiated cell, and can be said to be the primitive cell of all blood cells (most of which are immune cells). The content of the hematopoietic stem cells in the placenta tissue is 5-10 times of that in the umbilical cord blood, and the placenta tissue can be used by children for several times or used for 1-2 adult patients. Meanwhile, the technical problems of insufficient sources of bone marrow or mobilized peripheral blood, insufficient amount of umbilical cord blood and the like during transplantation are effectively solved, and the bone marrow, mobilized peripheral blood and umbilical cord blood are expected to be replaced for hematopoietic stem cell transplantation.
Decidua is a tissue formed by hypertrophy and proliferation of intimal stromal cells in the vicinity of a blastocyst of a mother in contact with the endometrium. Decidua basalis-forming placenta septa separate the dense chorion into several villous lobules, which contain maternal-derived decidua basalis stem cells.
Amnion (amnion) is a thin film covering the inner surface of amnion cavity, evolves from cell trophoblast, is the inner layer of human two-layer fetal membrane, has thickness of 0.02-0.05 mm, is the thickest basement membrane in human body, and consists of 5 layers of structures of amnion epithelial cell (HAEC), basement membrane, parenchymal layer, fibroblast layer and sponge layer. The human amniotic epithelial cells are positioned at the innermost layer of the amniotic membrane, and the amniotic epithelial cells are developed from the ectodermal leaves at the 8 th day after fertilization, so that the plasticity of embryonic stem cells at the prophase of gastral embryo formation can be maintained, and the application value in the aspect of treating central nervous system disorder is unique: in 1996, Sakuragawa et al first discovered that cultured human amniotic epithelial cells express markers for neurons and glial cells. Subsequent studies have shown that cultured human amniotic epithelial cells are capable of synthesizing and releasing neurotransmitters such as acetylcholine, catecholamines and dopamine. Experiments of treating rat Parkinson Disease (PD) by human amniotic epithelial cell transplantation show that the rat Parkinson Disease (PD) can survive and express the activity of the Tyrosine Hydroxylase (TH) in vivo, and can reverse the disease condition and prevent the death of neurons. The human amniotic epithelial cells also have good treatment effect on primate spinal cord injuries.
The umbilical cord is the ligament that connects the fetus to the mother. Three blood vessels pass through the umbilical cord, namely a vein and two arteries, and play a role in communicating blood circulation between mothers and children. The umbilical cord of a full-term fetus is about 30-70 cm long. The stem cells in Wharton's jelly are mainly umbilical cord mesenchymal stem cells.
Mesenchymal Stem Cells (MSCs) are pluripotent stem cells that are derived from the mesoderm during embryonic development. In the normal tissue damage repair process of the body, MSC is an important cell involved in tissue regeneration. Under the action of special signals caused by tissue damage, the MSCs migrate to the damaged part, gather and proliferate locally, and differentiate along different paths according to different damage signals. MSCs are easy to separate and amplify, have strong in vitro multiplication capacity, and can still maintain the multidirectional differentiation capacity even if the MSCs are amplified by 1 hundred million times. Thus, MSC is a practical tissue repair seed cell.
MSCs have a strong proliferative capacity and a multi-directional differentiation potential, and have the ability to differentiate into: epithelial stem cells, neural stem cells, hepatic stem cells, muscle cells, osteoblasts, chondrocytes, stromal cells, and the like. The MSC has the immunoregulation function, inhibits the immune response of organism hyperfunction through the negative immunoregulation function, and restores the balance of the immune function of the organism, thereby being used for treating the immune rejection reaction after the hematopoietic stem cell transplantation and autoimmune system diseases such as Crohn's disease, lupus erythematosus, scleroderma and the like; MSC is also an important component of a human body microenvironment, and transplantation of mesenchymal stem cells can change a hematopoietic microenvironment, rebuild an immune system, promote recovery of hematopoietic function, and remarkably improve the treatment effects of leukemia, refractory anemia and the like by being co-transplanted with hematopoietic stem cells.
The construction of a fetal accessory cell bank, wherein umbilical cord and placenta tissues of a newborn which are directly treated as medical wastes are treated to extract abundant stem cell resources, and the umbilical cord and placenta tissues are stored for a long time in a dormant state through a mature deep low temperature freezing technology and serve as precious biological resource reserves, so that the fetal accessory cell bank can be applied to the treatment of a plurality of autologous serious diseases in the future, and can also be applied to the transplantation and treatment of variant bodies; and the stem cell medicament can be developed through research, development and treatment, and the whole society is benefited.
The construction of the fetal accessory cell bank comprises the aspects of tissue separation, purification, cryopreservation, detection of pathogenic microorganisms, quality control and the like of cells. The method of constructing a fetal adjunct cell bank to which this patent relates is a method in which cells in a tissue are isolated, particularly a technique in which cells are isolated from a solid tissue. Methods for constructing cell banks by separating cells from tissues currently include chemical enzymatic hydrolysis and cell mass culture. Of which chemical enzymatic methods are the main methods widely used.
The chemical enzymatic method is a method of separating living cells from a tissue using a digestive enzyme. The specific separation process comprises shearing the tissue to about 1-1.5mm3The small blocks are digested by collagenase or a combination of collagenase and pancreatin, the digestion is stopped by enzyme digestion stop solution such as serum, and finally the cells are obtained by filtering and washing. For example, in patent No. CN101608174B, "a method for constructing a human umbilical cord mesenchymal stem cell bank", the mesenchymal stem cells are isolated by collagenase digestion. In patent No. CN100344757C, "human placenta and umbilical cord mesenchymal stem cell bank and its construction method", the mesenchymal stem cells in the bank are obtained by sequential digestion with collagenase and pancreatin. The chemical enzymolysis method has the following characteristics: 1) the experimental cost is high due to the need to use a large amount of chemical enzymes and digestion stop solutions, and 2) the cell yield obtained each time varies greatly due to different operating methods and personnel conditions. 3) Because of different digestion methods, the time of the whole digestion and enzymolysis is different from 30 minutes to several hours.
The cell mass culture method is a method for separating cells from tissues by combining physical and culture, and is an alternative method for constructing cell banks. The specific separation process comprises shearing the tissue to about 1-1.5mm3Inoculating the small blocks into a culture dish to culture and separate cells. For example, in patent No. CN102010850B, "a method for separating umbilical cord tissue mesenchymal stem cells by climbing sheet", the separation of umbilical cord tissue mesenchymal stem cells is a cell mass culture method. The method has the following characteristics: 1) it is only necessary to shear the tissue to 1-1.5mm3The cell mass and simple culture are simple, and the operation is simple; 2) the cell mass culture needs several days to realize the cell separation, which takes longer time, and 3) the long-term culture of the cells has higher possibility of pollution.
Therefore, the operation flow of the existing solid tissue cell separation method for constructing the cell bank is complicated, the time consumption is long, the method completely depends on manual operation, and the requirement on the operation skill of a technician is high. In addition, the chemical enzymolysis method introduces exogenous reagent of animal origin, which can increase the risk of stem cell drug therapy of public cell banks.
Based on the principle that the cell separation method is simple, time-saving, easy to control and standardize in the construction of the cell bank, the tissue homogenate method is adopted to separate the fetal accessory tissues to obtain a large number of cells for building the bank, so as to meet the requirements of the fields of scientific research, medicine, clinic and the like on cell bank resources.
Disclosure of Invention
The invention aims to provide a method for separating fetal accessory cells (clusters) by tissue homogenate to establish a cell bank.
The tissue cell processor PlacentaPro preferably used by the invention is an instrument designed by the inventor for the invention, and comprises a control device and a homogenizing container, as shown in figure 1, the control device comprises a machine body (1), a homogenizing container placing hole (2), a rotating head (3), a clamping groove (4) and a heat dissipation hole (5); the homogenate container comprises a cup body (6), a cup cover (7), a sealing ring (8), a rotary head connector (9), a fixed shaft (10), a cutter head (11) and a clamping head (12).
Fuselage (1) side is provided with louvre (5), hole (2) are placed for homogenate container at fuselage (1) top, homogenate container is placed hole (2) inner wall and is equipped with draw-in groove (4), homogenate container is placed hole (2) bottom and is rotating head (3), when handling the tissue, homogenate container is placed and is put into homogenate container and place hole (2), rotatory first connector (9) lower extreme and rotating head (3) joint of bowl cover (7) bottom, the upper end is passed through fixed axle (10) and is connected with tool bit (11), bowl cover (7) are through sealing washer (8) and cup body (6) close joint, dop (12) and draw-in groove (4) joint of cup body (6) lateral wall.
Wherein the key part is a specially designed tool bit (11). The cutter head (11) is a four-blade cutting edge and consists of a blade (13) and a blade (14) which are vertically stacked and perpendicular to each other. The blade (13) is divided into 5 sections and comprises 4 bends, two bends at two ends are at 130 degrees and 150 degrees (preferably 138 degrees), and two bends at the middle are at 140 degrees and 160 degrees (preferably 148 degrees); the blade (14) is divided into 3 sections and comprises 2 bends, and the two bends are 110 degrees and 130 degrees (preferably 123 degrees);
when the tissue homogenizer works, tissue blocks are placed into the cup body (6), after the cup cover (7) is covered, the homogenate container is placed into the homogenate container placing hole (2), and the transmission is carried out through a plurality of rows of the controller, the rotating head (3), the rotating head connector (9), the fixed shaft (10) and the cutter head (11). The cutter head (11) is driven to rotate, thereby achieving the effect of homogenizing.
Although a few instruments similar to the tissue cell processor PlacentaPro used in the present invention are currently on the market, such as the GentleMASC specimen processor from America and whirlwind, Germany, can also separate tissues to obtain cell suspensions. Firstly, the existing instruments on the market can only process samples with the processing capacity of less than 5g, and the PlacentaPro can process samples with the processing capacity of at most 400 g, so that a good foundation is laid for establishing a cell bank and culturing cells on a large scale, secondly, the existing instruments on the market can only process easily-broken tissues such as spleen, liver, lung and the like due to structural limitation, and cannot process tough fetal accessory tissues such as umbilical cord containing a large amount of Wharton's jelly, and the PlacentaPro can homogenize tough tissues such as fetal accessory tissues and directly separate cells/cell clusters without destroying the activity of the cells due to structural particularity.
The technical scheme of the invention comprises the processes of cleaning tissues, pre-homogenizing treatment, homogenizing tissues, filtering and recovering and constructing a cell bank, and the method comprises the following specific steps:
(1) tissue cleaning: the obtained fetal appendage tissue is washed by an appropriate amount of washing solution (e.g., PBS buffer containing double antibody) until the residual blood on the tissue surface is washed clean.
(2) Pretreatment of tissue homogenate: cutting the obtained tissue into small pieces according to the size of a homogenization container of PlacentaPro in a tissue cell processor, placing into the tissue cell processor, adding a buffer solution (such as PBS buffer solution) with 0.5-2 times of the mass, and sealing the tissue cell processor;
(3) tissue homogenization treatment: the tissue cell processor PlacentaPro cuts and homogenizes tissues by driving the special tool bit to rotate through the rotor, and separates and extracts cells of the tissues;
(4) tissue filtration treatment, cell collection: transferring the tissue fragments obtained in the step (3) to a filtering device, grinding the placenta tissue, collecting filtrate, cleaning the filtrate with cells, centrifuging to collect the cells, collecting cell clusters on the filter for the umbilical cord tissue, cleaning the cell clusters, and collecting.
(5) Isolated cells (clumps) cell banks were constructed: and (4) storing the cells (clusters) separated in the step (4) in liquid nitrogen, storing according to the ABO/Rh blood type and the HLA type of the cells, and establishing cell information files for retrieval, namely constructing a cell bank.
The sample to be treated according to step (1) of the present invention is a fetal appendage tissue including placental lobules and decidua basalis tissue, placental basement tissue, placental amniotic membrane tissue and umbilical cord tissue.
The tissue sample size treated according to step (2) of the present invention was:
30-120g, preferably 60-90g of placenta lobule and decidua basalis, and the volume of the added buffer solution or culture medium is 20-110ml, preferably 50-80 ml;
5-30g, preferably 10-20g of umbilical cord, and the volume of the added buffer solution or culture medium is 10-45ml, preferably 15-30 ml;
the placenta base is 100-300g, preferably 150-200g, and the volume of the added buffer solution or culture medium is 100-300m1, preferably 150-200ml;
5-25g, preferably 10-20g of placenta amnion, and the volume of the added buffer solution or culture medium is 10-40ml, preferably 15-25 ml;
the tissue cell processor plantapro treatment according to step (2) of the present invention:
the treatment rotating speed of the placental lobules and decidua basalis tissues is 1000-2400rpm, preferably 1400-1800 rpm;
the umbilical cord tissue treatment rotating speed is 1500-;
the placenta base tissue processing rotating speed is 1500-;
the processing rotating speed of the placental amniotic membrane tissue is 2000-3000rpm, preferably 2300-2500 rpm;
the tissue fragment filtering device in the step (4) of the invention is a 100-mesh 400-mesh metal filter, and the filter with 200 meshes is preferably used for filtering the placenta lobule, decidua basalis tissue and the placenta base tissue, and the filter with 400 meshes is preferably used for filtering the umbilical cord tissue and the placenta amnion tissue.
The invention has the beneficial effects that: the method for separating the fetal accessory histiocytes by adopting the independently developed histiocyte processor PlacentaPro has the advantages of simple operation, short time, no addition of exogenous reagents, easy standardization, high quality control, large cell acquisition quantity and the like, and has important significance for improving the construction efficiency of the cell bank and ensuring the high-quality of the cell bank.
Drawings
FIG. 1 schematic diagram of the structure of a tissue cell processor PlacentaPro
FIG. 2 Primary cell culture morphology
FIG. 3 morphology of cell cultures from umbilical cord tissue treatment in cell clumps cultured and transferred to P3 passages
FIG. 4 cell flow diagram of cell mass culture from umbilical cord tissue treatment and passage to P3
FIG. 5 Primary culture morphology of cell clusters obtained after placental amniotic membrane tissue treatment
Detailed Description
The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1: cell bank constructed by separating placenta lobular cells by tissue homogenate method
The method for homogenizing the tissue of the placenta lobule comprises the following steps:
(1) cleaning placenta tissues: the placenta tissue is treated in a biological safety cabinet, and a proper amount of PBS (phosphate buffer solution) containing 1% double antibody is used for washing the placenta tissue according to the size of the placenta until residual blood on the surface of the placenta tissue is washed clean and no blood clot exists on the surface of the placenta tissue;
(2) and (3) placenta lobule tissue prophase treatment: cutting off placental leaflets from the placental tissue obtained in the step (1) by using a surgical scissors, transferring the placental leaflets onto a culture dish and cutting the placental leaflets into small pieces, putting 60-90g of placental leaflet tissue pieces into a homogenization container of a histiocyte processor PlacentaPro, adding 50-80ml of PBS and sealing a processing cup;
(3) homogenating the placenta lobule tissue: separating and extracting cells of the placenta lobular tissue block obtained in the step (2) by physical treatment of tissue cutting and homogenizing by using a tissue cell processor PlacentaPro, wherein the stirring speed is 1400-1800rpm, and the treatment time is 5 minutes;
(4) and (3) filtering placenta lobule tissues: transferring the placenta lobule tissue treated in the step (3) to a 200-mesh metal filter, grinding the tissue fragments, collecting the filtered liquid by using another culture dish, adding 10-15ml of PBS into the metal filter for washing the tissue in 2 times, and continuously grinding.
(5) Cell counting: the cell suspension obtained in step (4) was collected in a 50ml centrifuge tube, centrifuged at 1400 rpm/min for 10 minutes, the supernatant was removed and PBS was added to resuspend the cells, and a small sample was withdrawn for cell counting.
The placental lobule histiocyte isolation treatment test was performed in 8 separate groups, each with two different test settings, respectively, (a)1400rpm, 5 minutes and (b)1800rpm, 5 minutes. Placental tissue was derived from 8 different donors and the treatment was between about 60 grams. The results of the experiment are shown in table 1.
TABLE 1(a) placental lobule tissue homogenate cell extraction data
TABLE 1(b) placental lobule tissue homogenate processing cell extraction data
From the above results, it can be seen that the placenta lobule tissue was treated by the fully automatic placenta organizer under the test conditions of (a)1400rpm, 5 minutes and (b)1800rpm, 5 minutes, and the average number of nucleated cells per gram was 2.98 × 106And 2.8X 106And the cell quantity obtained by the two methods is not obviously different, so that the improvement of the speed has no clear linear relation on the cell extraction efficiency. Moreover, in a supplementary experiment, the cells in which the cell surface marker CD34 was positively expressed were found to be 2.92X 10 cells, respectively, by flow cytometry4G and 2.78X 104In another supplementary experiment, improper homogenization times and rotation rates that were too high and too low affect the efficiency of cell extraction. The survival rate test shows that the nucleated cells separated and obtained by the placenta tissue processor still keep good activity, and the survival rate of 8 test results can reach more than 98%. The above results show that the fully automatic tissue cell treatmentThe device, PlacentaPro, can effectively separate and extract nucleated cells from placenta lobular tissue, is simple and easy to implement, consumes short time, and provides effective guarantee for the construction of a cell bank.
Example 2: comparative experiment of placenta lobule tissue homogenate treatment and manual treatment
The comparative experiments were performed in 8 groups. A fully automated tissue processing experiment was performed with reference to the method of example 1. The manual processing (digestion) experiments were carried out with reference to the method described in patent CN201210292509.9 (publication No. CN 102807966A). Placental tissue was derived from 8 different donors at treatment volumes between 60-90 grams. The results of the comparative experiments are shown in table 2.
| Fully automated processing | Manual processing (digestion) | |
| Testing | Number of extracted cells/g | Number of extracted cells/g |
| 1 | 3.8×106／g | 2.0×106／g |
| 2 | 3.9×106／g | 0.79×106／g |
| 3 | 2.3×106／g | 1.0×106／g |
| 4 | 2.9×106／g | 1.1×106／g |
| 5 | 2.6×106／g | 1.4×106／g |
| 6 | 2.7×106／g | 1.1×106／g |
| 7 | 3.1×106／g | 0.86×106／g |
| 8 | 2.5×106／g | 1.7×106／g |
| Average | 2.98×106／g | 1.24×106／g |
TABLE 2 comparison table of fully automatic and manual treatment of placental lobule tissue data
The result shows that the average cell amount per gram obtained after the placenta lobular tissue is treated by a full-automatic tissue cell processor PlacentaPro is 2.4 times of that obtained by manual treatment, and is obviously higher than that obtained by manual digestion treatment, a large number of cells are provided for establishing a cell bank, and the time for subsequent cell expansion is saved.
Example 3: primary cell growth from placental lobule tissue isolated by homogenization
(1) Preparation before cell culture: adding erythrocyte lysate (Roche) according to the proportion of 1 part of cell suspension to 2-3 parts of erythrocyte lysate, culturing for 10-15 minutes at 15-25 ℃, putting into a centrifuge, centrifuging for 10 minutes at 1400rpm, removing supernatant, observing erythrocyte lysis condition, and repeating the step for lysis if necessary. After lysis, add PBS to resuspend the cells and draw a small sample for cell counting, place in centrifuge to wash cells by centrifugation at 1400rpm for 10 minutes, remove supernatant, add mesenchymal stem cell medium (15% FBS + 1% L-Glutamine + 0.05% Gentamicin + 84% DMEM-F12) to resuspend the cells, and add 2-5X 104／cm2The placenta-derived nucleated cells are inoculated into culture flasks.
(2) Primary cell culture: placing the culture flask into C02Culturing in an incubator with concentration of 5% and temperature of 37 deg.C, performing first half-change when culturing to day 6, performing second half-change on day 9, removing culture medium in the plate on day 12, adding 15ml culture medium again, and performing total change every 2-3 days later. The cell culture form is observed by a microscope, the adherent growth of the cells is in a typical fusiform shape, the cytoplasmic prominence is in a fibroblast shape, the cytoplasm is rich, the nucleolus is obvious, and the cells grow in a vortex shape. (see FIG. 2)
(3) Cell passage: when the fusion rate of adherent cells in the culture bottle reaches about 80%, detaching the adherent cells from the bottom of the culture bottle by utilizing digestive enzyme (trypLEexpress), centrifuging, then pumping out the supernatant, adding the suspended cells into a special cell culture medium, inoculating the suspended cells into the cell culture bottle for passage and continuous amplification culture, and changing the liquid every 2 days until the fusion rate reaches 80%, thus obtaining the culture medium, and if necessary, carrying out passage again.
The primary culture was performed on 8 groups of cells extracted in example 3, and the growth results are shown in table 3.
TABLE 3 Primary growth of isolated cells from placental lobules
The 8 groups of placental lobules are subjected to the same culture conditions of nucleated cells obtained by separating the placental lobules by a tissue cell processor PlacentaPro, and the primary growth states of the cells of each group are basically consistent and the cells are full of primary cells in about 12-14 days. The consistency shows that the full-automatic homogenization method has small difference among samples, high standardization degree and higher quality assurance when large-scale sample treatment is carried out.
Example 4: separation of umbilical cord cell mass by tissue homogenization
The umbilical cord tissue homogenizing method comprises the following steps:
(1) cleaning umbilical cord tissues: the umbilical cord tissue is treated in a biological safety cabinet, the umbilical cord tissue is cut open, blood vessels in the umbilical cord are taken out, then the umbilical cord tissue is washed by PBS buffer solution containing 1% double antibody until residual blood on the surface of the umbilical cord tissue is washed clean and no blood clot exists on the surface of the umbilical cord;
(2) pre-treatment of umbilical cord tissue: transferring the umbilical cord tissue obtained in the step (1) to a culture dish, shearing the umbilical cord tissue into 2cm by 2cm small pieces by using a surgical scissors, putting 10-20g of umbilical cord tissue pieces into a homogenization container of a tissue cell processor PlacentaPro, adding 20-30 ml of 1% double-antibody-containing PBS, and sealing the homogenization container;
(3) homogenizing umbilical cord tissues: separating and extracting cell mass of the umbilical cord tissue obtained in the step (2) by physical treatment of tissue cutting and homogenization by using a tissue cell processor PlacentaPro, wherein the stirring speed is 2500-;
(4) umbilical cord tissue filtration treatment: transferring the umbilical cord tissue treated in the step (3) to a 400-mesh metal filter, collecting cell clusters on the filter, and dividing the cell clusters into 2 times of washing the cell clusters with 5ml of physiological saline.
(5) Carrying out adherent culture on the primary cells by using umbilical cord cell masses: spreading the cell mass obtained in the step (4) in a plate, adding a mesenchymal stem cell culture medium (15% FBS + 1% L-Glutamine + 0.05% Gentamicin + 84% DMEM-F12), and placing the plate in a CO container2Culturing in a 5% culture box at 37 deg.C until 6 days, performing the first half-change, performing the second half-change on 9 days, and performing the whole-change every 2-3 days from 12 days. The cell culture form is observed by a microscope, the adherent growth of the cells is in a typical fusiform shape, the cytoplasmic prominence is in a fibroblast shape, the cytoplasm is rich, the nucleolus is obvious, and the cells grow in a vortex shape.
(6) Cell passage: when the fusion rate of adherent cells in the plate reaches about 80%, detaching the adherent cells from the bottom of the culture bottle by utilizing digestive enzyme (trypLEexpress), centrifuging, then pumping out the supernatant, adding the suspended cells into a special cell culture medium, inoculating the suspended cells into the cell culture bottle for passage and continuous amplification culture, and changing the liquid every 2 days until the fusion rate reaches 80%, thus obtaining the culture medium, and if necessary, carrying out passage again. FIG. 3 is a morphological diagram of the cell mass obtained from the umbilical cord tissue treatment and transferred to the P3 generation, and FIG. 4 is a flow chart of the cell mass obtained from the umbilical cord tissue treatment and transferred to the P3 generation.
Example 5: placenta base cell separation by tissue homogenate method
The method for homogenating placenta base tissue includes the following steps:
(1) cleaning placenta tissues: the placenta tissue is treated in a biological safety cabinet, and is washed for 2-3 times by using a proper amount of PBS (phosphate buffer solution) according to the size of the placenta, so that residual blood on the surface of the placenta tissue is washed clean, and blood clots do not exist on the surface of the placenta;
(2) and (3) placenta base tissue pretreatment: cutting off a placenta base from the placenta tissue obtained in the step (1) by using a surgical scissors, transferring the placenta base onto a culture dish and cutting into small pieces, putting the placenta base tissue pieces between 150 and 200g into a homogenate container of a tissue cell processor PlacentaPro, adding 150 and 200ml of PBS and sealing the homogenate container;
(3) homogenating the placenta base tissue: separating and extracting cells of the placenta base tissue obtained in the step (2) by physical treatment of tissue cutting and homogenizing by using a tissue cell processor, namely PlacentaPro, wherein the stirring speed is 2000rpm, and the treatment time is 5 minutes;
(4) and (3) filtering the placenta base tissue: transferring the placenta base tissue treated in the step (3) to a 200-mesh metal filter, grinding tissue fragments, collecting filtered liquid by using another culture dish, adding 20-25ml of PBS into the metal filter for washing the tissue in 2 times, and continuously grinding.
(5) Cell counting: the cell suspension obtained in step (4) was collected in a 50ml centrifuge tube, centrifuged at 1400 rpm/min for 10 minutes, the supernatant was removed and PBS was added to resuspend the cells, and a small sample was withdrawn for cell counting.
The placental base tissue cell separation treatment test was carried out for 8 groups, respectively, under the test conditions of 2000rpm for 5 minutes. Placental tissue was derived from 8 different donors at a throughput of between 150 and 200 grams. The results of the experiment are shown in table 4.
Table 4 placental base tissue homogenization treatment cell extraction data
From the above results, it can be seen that the placental cell processor, plantenaPro, treated the placenta mount tissue under the test conditions of 2000rpm for 5 minutes, and finally obtained the average number of nucleated cells per gram of 6.1X 105. In supplementary experiments, improper homogenization times and rotation rates that are too high and too low can affect the efficiency of cell extraction. The survival rate test shows that the nucleated cells separated and obtained by the tissue cell processor still keep good activity, and the survival rate of 8 test results can reach more than 99%. The results show that the fully automatic histiocyte processor PlacentaPro can effectively separate and extract nucleated cells from placenta base tissues, is simple and easy to implement, consumes short time and provides effective guarantee for the construction of a cell bank.
Example 6: separation of placental amniotic cells by tissue homogenization
The placental amniotic membrane tissue homogenization method comprises the following steps:
(1) cleaning placenta tissues: the umbilical cord tissue is treated in a biological safety cabinet, and is washed for 2-3 times by using a proper amount of PBS (phosphate buffer solution) according to the size of the umbilical cord, so that residual blood on the surface of the umbilical cord tissue is washed clean, and blood clots do not exist on the surface of the umbilical cord;
(2) and (3) placenta amniotic membrane tissue prophase treatment: cutting off the placental amniotic membrane from the placental tissue obtained in the step (1) by using a surgical scissors, transferring the amniotic membrane tissue to a culture dish and cutting into small pieces, putting 10-20g of placental amniotic membrane tissue pieces into a homogenization container of a tissue cell processor PlacentaPro, adding 15-25ml of PBS, and sealing the homogenization container;
(3) homogenizing the placenta amnion tissue: separating and extracting cells of the placental amniotic membrane tissue obtained in the step (2) by physical treatment of tissue cutting and homogenization by using a tissue cell processor, namely PlacentaPro, wherein the stirring speed is 2300-2500rpm, and the treatment time is 8 minutes;
(4) and (3) filtering the placenta amniotic membrane tissue: transferring the placenta amnion tissue treated in the step (3) to a 400-mesh metal filter, collecting cell clusters on the filter, and washing the cell clusters with 5ml of PBS buffer solution for 2 times.
(5) Primary cell culture: adding amniotic epithelial cell culture medium (10% FBS + 90% DMEM +10 ng/ml EGF +4mmo 1/L glutamine) to the cell mass obtained in step (5), and placing the plate in a C02Culturing in an incubator with concentration of 5% and temperature of 37 deg.C, performing first half-change of culture solution until day 6, performing second half-change of culture solution at day 9, and performing total change of culture solution every 2-3 days from day 12. The morphology of the cell culture was observed by microscopy and the cells grew adherently as plump cobblestones, as shown in fig. 5.
Example 7: cryopreservation of cells (clusters) separated by fetal accessory tissue homogenate method
Cells/cell clusters homogenized, filtered and washed by a tissue cell processor can be frozen and stored at a deep low temperature if the cells/cell clusters are temporarily inapplicable, and then taken out for recovery culture when needed later.
The cryopreservation method of the placental cells comprises the following steps:
take 1X 106Adding the cells into 1ml of cell frozen stock solution (containing 65 parts of DMEM-F12, 10 parts of dimethyl sulfoxide and 15 parts of human serum albumin), performing programmed cooling for freezing treatment, and finally putting the frozen sample into a liquid nitrogen tank for freezing.
The cryopreservation method of the umbilical cord cell mass comprises the following steps:
adding 1ml of cell cryopreservation liquid (containing 80 parts of human serum albumin and 20 parts of dimethyl sulfoxide) into a cryopreservation tube, adding the umbilical cord cell mass into the cryopreservation liquid, wherein the total volume is not more than 1.8ml, performing programmed cooling for freezing treatment, and finally putting the frozen sample into a liquid nitrogen tank for cryopreservation.
Example 8 establishment of fetal Accessory cell (Cluster) library
1. Investigation of cell origin
Details of the placenta provider (parent) were recorded and documented.
2. Detection of cell contamination
And (3) detecting whether the cells are polluted by fungi and bacteria by using a small amount of cell culture. And (3) detecting whether the cells are infected by hepatitis B two-and-two, hepatitis C, AIDS, cytomegalovirus, EB virus, syphilis, HbsAg, HbsAb, HBcAb, HbeAg, HbeAb, HCVAb, HIV-1/2 Ab, CMV-IgM, EBV-IgA and TRUST by utilizing an etiological method.
3. Detection of genetic disorders
And detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
4. HLA-ABC/DR match
Cells were tested for HLA-ABC/DR phenotype and recorded.
5. Detection of cellular Activity
The number of viable cells before and after cryopreservation was counted using trypan blue staining.
6. Establishment of placental stem cell database
After normal placental stem cells are preserved, a database of placental stem cells is established, which includes the first five items of information, and associations with cryopreserved cells are established.
Claims (5)
1. A method for constructing a cell bank by tissue homogenate method for fetal accessory tissue isolation, which comprises the following steps:
(1) carrying out ABO/Rh blood type detection, HLA type detection and microorganism immunity detection on a fetal accessory tissue provider, and removing residual blood by using a buffer solution containing double antibodies, wherein the fetal accessory tissue is a placenta base;
(2) pretreatment of tissue homogenate: cutting the cleaned tissue into small pieces, placing the small pieces into a sample chamber of a tissue cell processing device, adding a buffer solution or a culture medium with the mass of 0.5-2 times that of the small pieces, and sealing the tissue cell processing device;
(3) tissue homogenization treatment: operating the tissue cell processing device, cutting and homogenizing the tissue under the conditions of specified rotating speed and time, and separating the cells of the tissue; wherein the processing speed is 2000rpm, and the processing time is 5 minutes;
(4) tissue filtration treatment: filtering the homogenate obtained in the step (3) through a 200-mesh filter screen: for placenta base tissues, grinding a few placenta tissue blocks remained on the filter screen, and collecting filtrate;
(5) cell collection and washing: for cells, collecting the cell suspension obtained in the step (4) by using a centrifuge tube, centrifuging at the speed of 1400-1800 rpm/min for 5-15 min, removing supernatant, and adding a buffer solution to resuspend the cells;
(6) preserving the cells separated in the step (5) in liquid nitrogen, preserving according to ABO/Rh typing and HLA typing of the cells, establishing cell information files for retrieval, namely establishing a cell bank,
wherein,
the tissue cell processing device is a tissue cell processor which comprises two parts of a control device and a homogenizing container, wherein:
the control device comprises a machine body (1), a homogenate container placing hole (2), a rotating head (3), a clamping groove (4) and a heat dissipation hole (5);
the homogenate container comprises a cup body (6), a cup cover (7), a sealing ring (8), a rotary head connector (9), a fixed shaft (10), a tool bit (11) and a chuck (12);
the novel tissue treatment machine is characterized in that heat dissipation holes (5) are formed in the side face of the machine body (1), a homogenate container placing hole (2) is formed in the top of the machine body (1), a clamping groove (4) is formed in the inner wall of the homogenate container placing hole (2), a rotating head (3) is arranged at the bottom of the homogenate container placing hole (2), during tissue treatment, the homogenate container is placed in the homogenate container placing hole (2), the lower end of a rotating head connector (9) at the bottom of a cup cover (7) is connected with the rotating head (3), the upper end of the rotating head connector is connected with a tool bit (11) through a fixed shaft (10), the cup cover (7) is tightly connected with a cup body (6) through a sealing ring (8), and a clamping;
the cutter head (11) is a four-blade cutting edge and consists of a first blade (13) and a second blade (14) which are vertically stacked and perpendicular to each other; the first blade (13) is divided into 5 sections, which comprise 4 bends, two bends at two ends are at 150 degrees and two bends at the middle are at 160 degrees and 140 degrees; the second blade (14) is divided into 3 sections and comprises 2 bends, and the two bends are 110 DEG and 130 DEG;
when the histiocyte processing device works, tissue blocks are placed into the cup body (6), after the cup cover (7) is covered, the homogenate container is placed into the homogenate container placing hole (2), and the cutter head (11) is driven to rotate through a series of transmission of the controller, the rotating head (3), the rotating head connector (9), the fixed shaft (10) and the cutter head (11), so that the homogenate effect is achieved.
2. The method according to claim 1, wherein in the step (2), the placenta basement tissue treated by the tissue cell treating device is 100-300 g.
3. The method of claim 1, wherein in step (6), the cell bank is selected from the group consisting of: placenta hematopoietic stem cell bank, placenta sub totipotent cell bank.
4. The method of claim 1, wherein in step (6), said creating a cell information file available for retrieval comprises a survey of cell origin; detecting cell contamination; detecting a genetic disease; HLA-ABC/DR match detection; and (4) detecting the activity of the cells.
5. The method of claim 4, wherein the detection of cell contamination comprises detection of: hepatitis B, hepatitis C, AIDS, cytomegalovirus, EB virus and syphilis.
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| CN104480533A (en) * | 2014-12-29 | 2015-04-01 | 黑龙江天晴干细胞股份有限公司 | Placenta stem cell bank construction method and placenta tissue resuscitation method |
| JP2019528728A (en) * | 2016-09-29 | 2019-10-17 | 高雄醫學大學Ｋａｏｈｓｉｕｎｇ Ｍｅｄｉｃａｌ Ｕｎｉｖｅｒｓｉｔｙ | Device for separating cells from tissue |
| CN106399237A (en) * | 2016-11-30 | 2017-02-15 | 广州赛莱拉干细胞科技股份有限公司 | Method for primary isolation of umbilical cord mesenchymal stem cells |
| CN106801032B (en) * | 2017-02-17 | 2021-03-02 | 沈阳艾米奥生物工程技术研发中心有限公司 | Construction method of human amniotic epithelial stem cell bank |
| CN107541485A (en) * | 2017-10-19 | 2018-01-05 | 四川农业大学 | A kind of original, primary, secondary follicle the separation method of goose |
| CN108728408B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Canine fetal membrane mesenchymal stem cells and preparation method and medium used |
| CN108795853B (en) * | 2018-05-28 | 2021-08-24 | 天津博雅秀岩生物技术有限公司 | Method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells |
| CN110859856A (en) * | 2019-10-21 | 2020-03-06 | 中冠赛尔生物科技(北京)有限公司 | Transplant for reducing GVHD morbidity |
| CN112506620B (en) * | 2020-12-28 | 2023-11-24 | 网络通信与安全紫金山实验室 | Cleaning and recovery methods, devices, equipment and media based on docker container deployment of ospf protocol |
| CN114958717B (en) * | 2022-04-06 | 2023-03-10 | 中山大学附属第一医院 | Method for quickly separating living cells from trace calcified blood vessels |
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