Cryopreservation liquid and method for cryopreservation and resuscitation of umbilical cord tissue blocks, preparation of mesenchymal stem cells and establishment of seed bank

Abstract

The invention relates to the technical field of biology, in particular to a cryopreservation solution and a method for cryopreservation and resuscitation of umbilical cord tissue blocks, preparation of mesenchymal stem cells and establishment of a seed bank.The cryopreservation solution is prepared from 85% of a serum-free medium, 5% of platelet lysate and 10% of DMSO. After being cooled according to a program, the tissue blocks are stored in a liquid nitrogen deep hypothermia environment, the mesenchymal stem cells are cultured after the tissue blocks are recovered, the growth state of the cells can be obviously improved through serum-free culture, and differentiation osteogenesis experiments and differentiation adipogenesis experiments are carried out on the cells cultured by the cryopreserved tissue blocks, so that the differentiation function is very good. The method provided by the invention can effectively reduce waste of stem cell resources and damage to cells. According to the method, umbilical cord tissue blocks can be stored for a long time, nutrient substances required by cell growth are effectively provided, the condition of poor cell growth state is improved, and the umbilical cord tissue blocks can be recovered and amplified according to needs and then applied to treatment of future diseases.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a cryopreservation solution and a method for cryopreserving and thawing umbilical cord tissue blocks, preparing mesenchymal stem cells and establishing a seed bank. Background Art

[0002] The umbilical cord is a cord-like or tubular structure that connects the fetus to the maternal placenta during the fetal period. It is composed of two arteries and one vein. Between the arteries and veins of the umbilical cord is a special embryonic mucous connective tissue - Wharton's jelly. Wharton's jelly is a gel-like substance that makes up the umbilical cord. It is a mucosal tissue that contains mucopolysaccharides, fibroblasts and macrophages. The preparation of human umbilical cord mesenchymal stem cells mainly comes from Wharton's jelly tissue, and the umbilical cord Wharton's jelly is very rich in MSC. Mesenchymal stem cells (MSCs) are a branch of stem cells that have the ability to self-replicate and multidirectionally differentiate. They can continuously renew themselves and transform into one or more cells that constitute human tissues or organs under specific conditions. Their application in the field of cell therapy is becoming more and more extensive, and significant clinical effects have been achieved in the fields of organ damage diseases, nervous system diseases, endocrine system diseases, etc.

[0003] Umbilical cord-derived MSCs have the characteristics of wide sources, easy collection, relative purity, very rich content and low immunogenicity. Therefore, the preservation of umbilical cord-derived MSCs has received increasing attention.

[0004] At present, the general practice of preserving umbilical cord stem cells in the industry is to first separate mesenchymal stem cells from Wharton's jelly, and then freeze them after amplification and culture to a certain number, and store them in liquid nitrogen at -196°C. However, this method has a long cycle, about 20 days of culture, and high preparation costs. In addition, after the preparation of mesenchymal stem cells, the Wharton's jelly tissue blocks are discarded as waste, resulting in a waste of stem cell resources. The tissue blocks can be stored in a 4°C refrigerator for up to 7 days. If the cells are cultured after more than 7 days, it will be difficult for the cells to grow. In addition, the serum-containing culture medium is used when preparing the umbilical cord tissue blocks. After the tissue blocks are revived, the cells are cultured, the cell state is poor, and the ability to adhere to and grow primary cells is insufficient. In addition, the heterologous contamination caused by animal serum can bring risks to the application of clinical-grade MSCs.

[0005] In addition, currently most umbilical cord tissue cryopreservation solutions use a mixture of serum culture medium and DMSO. For example, the cryopreservation solution used for tissue block cryopreservation in patent CN104472473B contains serum. Although this solves the problem of umbilical cord tissue preservation time, when necessary, the tissue block is revived and the MSC is cultured in serum, which has poor cell status and is prone to heterologous contamination. Summary of the invention

[0006] One of the purposes of the present invention is to provide a freezing solution that can effectively improve the cell state of umbilical cord tissue blocks after resuscitation in view of the deficiencies in the prior art.

[0007] The second purpose of the present invention is to provide a simple, efficient and pollution-free method for cryopreservation, resuscitation and preparation of mesenchymal stem cells of umbilical cord tissue blocks in view of the deficiencies in the prior art. The third purpose of the present invention is to provide a method for establishing an umbilical cord mesenchymal stem cell seed bank in view of the deficiencies in the prior art.

[0008] The purpose of the present invention is achieved through the following technical solutions: Technical solution 1: A freezing solution, calculated by volume percentage, is a mixture of the following components: α-MEM medium: 80-90% Platelet lysate: 4-6% Dimethyl sulfoxide: 8-12%.

[0009] Preferably, the freezing solution consists of the following ingredients: α-MEM medium: 85% Platelet lysate: 5% Dimethyl sulfoxide: 10%.

[0010] Technical Solution 2: A method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks, which comprises the following steps in sequence: Step (1) Umbilical cord collection: immerse the umbilical cord in DPBS solution and transport it to the laboratory using a biosafety transport box; Step (2) cleaning the umbilical cord: disinfect the collected umbilical cord with a disinfectant, clean it with a DPBS solution, cut it into tissue segments, and wash it again with a DPBS solution until the tissue segments turn white; Step (3) separation of Wharton's jelly: use ophthalmic forceps to separate the Wharton's jelly from the tissue segment and cut it into tissue blocks, and wash it with DPBS solution; Step (4) Cryopreservation of tissue blocks: adding a cryopreservation solution to the tissue blocks, wherein the volume of the cryopreservation solution added is one-half of the volume of the tissue blocks, and dispensing the solution into cryopreservation tubes. The cryopreservation tubes are placed in a programmed cooling cryopreservation box, and are successively refrigerated at -4°C for 2 hours, frozen at -80°C overnight, and finally frozen in liquid nitrogen at -196°C for later use; Step (5) tissue block recovery: when necessary, take out the frozen tissue block in step (4) from liquid nitrogen, thaw it in a 37°C constant temperature water bath, put it into a 15 ml centrifuge tube, add 5 times the volume of serum-free culture medium, mix and centrifuge, and remove the supernatant; Step (6) Use a pipette to transfer the tissue blocks revived in step (5) into a culture bottle containing serum-free medium and incubate at 37°C and 5% CO 2 , cultured in an incubator with saturated humidity; Step (7) On the 5th to 7th day after culture, the medium is fully replaced, and then half the medium is replaced every 3 days until the cells crawl out from the tissue block. After the cell confluence reaches more than 70%, digestion solution is used for digestion and passage, thereby obtaining umbilical cord mesenchymal stem cells, which are recorded as P0 cells.

[0011] Preferably, the DPBS solution is a double-antibody DPBS buffer, that is, it contains streptomycin and penicillin accounting for 1% of the volume of the DPBS solution.

[0012] Preferably, the disinfectant is alcohol with a volume concentration of 75%.

[0013] Preferably, the length of the tissue segment is 3-4 cm; the volume of the tissue block is 0.9-1.1 mm 3 , 0.9-1.1mm3 tissue blocks, effectively preventing the formation of ice crystals in the intracellular fluid, thereby reducing damage to the cells.

[0014] Preferably, the freezing solution is prepared by mixing 85% α-MEM culture medium, 5% platelet lysate, and 10% dimethyl sulfoxide in a 50 ml centrifuge tube in a volume ratio and precooling in a 4° C. refrigerator.

[0015] Preferably, the serum-free medium consists of α-MEM and platelet lysate. More preferably, the following ingredients are specifically included (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0016] Preferably, the digestive fluid is pancreatic digestive fluid.

[0017] Furthermore, it also includes: Step (8) Inoculate the P0 cells into a T25 flask at a cell density of 1E5, add 5 ml of serum-free medium, and place at 37°C and 5% CO 2 , static culture in an incubator with saturated humidity; Step (9) When the cell confluence reaches 70-80%, the cells can be subcultured. The serum-free medium supernatant is removed, 2 ml of DPBS solution is added to wash away the excess serum-free medium, which is then discarded. 1 ml of 0.25% trypsin digestion solution is taken and digested for 2 min. Two times the volume of the digestion solution is added to terminate the digestion. The cells are transferred to a centrifuge bottle tube and centrifuged. The supernatant is discarded to obtain P1 cells.

[0018] Technical Solution 3: A method for establishing an umbilical cord mesenchymal stem cell seed bank, the method comprising the following steps: (1) Record the fetal information and the genetic virus detection information of the donor maternal blood in the collected umbilical cord; (2) After the obtained umbilical cord mesenchymal stem cells are passaged, cells from passages 3 to 6 are taken for cell biological property testing, stability testing, safety testing, and cell differentiation potential testing, and all relevant test data of the cells are retained; (3) After all the data are qualified, an umbilical cord mesenchymal stem cell seed bank is established. The cells are stored in liquid nitrogen at -196°C and revived and expanded when needed for use in cell therapy.

[0019] Beneficial effects of the present invention: The cryopreservation solution of the present invention is prepared by 85% serum-free culture medium, 5% platelet lysate and 10% DMSO. The tissue block and the cryopreservation solution are added to the cryopreservation tube, and the cryopreservation tube is stored in a liquid nitrogen deep cryogenic environment after being cooled according to the program. The mesenchymal stem cells are cultured after the tissue block is revived. The serum-free culture can significantly improve the cell growth state, and the cells cultured in the frozen tissue block are subjected to osteoblastic differentiation experiments and adipogenic differentiation experiments, and the differentiation function is very good. The method of the present invention can effectively reduce the waste of stem cell resources and damage to cells. The method of the present invention can be applied to the preservation of the umbilical cord after the birth of a newborn. The umbilical cord tissue block can be stored for a long time in a liquid nitrogen deep cryogenic environment and has activity, growth ability and differentiation ability similar to fresh tissue. The method of the present invention can preserve the umbilical cord tissue block for a long time, which effectively provides the nutrients required for cell growth, improves the poor cell growth state, and can be revived and amplified as needed and applied to the treatment of future diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Prior art serum-containing cell culture diagram; Figure 2 This is a diagram of serum-free cultured mesenchymal stem cells obtained in Example 4; Figure 3 This is a picture of serum-free cultured P1 generation cells obtained in Example 4; Figure 4 This is a diagram of osteogenic differentiation of mesenchymal stem cells obtained in Example 4; Figure 5 This is a diagram of the adipogenic differentiation of the mesenchymal stem cells obtained in Example 4. DETAILED DESCRIPTION

[0021] The present invention is further described in conjunction with the following examples. Embodiment 1

[0022] A freezing solution, calculated by volume ratio, the freezing solution consists of the following components: α-MEM medium: 82% Platelet lysate: 6% Dimethyl sulfoxide: 12%, The above components are mixed evenly in a centrifuge tube according to proportion and precooled in a refrigerator at 4°C. Embodiment 2

[0023] A freezing solution, calculated by volume ratio, the freezing solution consists of the following components: α-MEM medium: 85% Platelet lysate: 5% Dimethyl sulfoxide: 10%, The above components are mixed evenly in a centrifuge tube according to proportion and precooled in a refrigerator at 4°C. Embodiment 3

[0024] A freezing solution, calculated by volume ratio, the freezing solution consists of the following components: α-MEM medium: 88% Platelet lysate: 4% Dimethyl sulfoxide: 8%, The above components are mixed evenly in a centrifuge tube according to proportion and precooled in a refrigerator at 4°C. Embodiment 4

[0025] A method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks, comprising the following steps in sequence: For umbilical cord collection, the umbilical cord was soaked in DPBS solution and transported to the laboratory in a biosafety transport box; Umbilical cord cleaning: the collected umbilical cords are disinfected with disinfectant, cleaned with DPBS solution, cut into tissue segments, and washed again with DPBS solution until the tissue segments turn white; Separation of Wharton's jelly: Use ophthalmic forceps to separate the Wharton's jelly from the tissue segment and cut it into tissue blocks, and then wash it with DPBS solution; For tissue block freezing, add freezing solution to the tissue block, the volume of the added freezing solution is half of the volume of the tissue block, and dispense into cryotubes. Place the cryotubes in a programmed cooling cryobox, refrigerate at -4°C for 2 hours, freeze at -80°C overnight, and finally freeze in liquid nitrogen at -196°C for later use; Tissue block recovery: When necessary, take out the frozen tissue block in step (4) from liquid nitrogen, thaw it in a 37°C constant temperature water bath, put it into a 15 ml centrifuge tube, add 5 times the volume of serum-free culture medium, mix well, centrifuge at 1000r for 5 min, and remove the supernatant; Use a pipette to transfer the tissue blocks revived in step (5) to a culture bottle containing serum-free medium and incubate at 37°C and 5% CO 2 , after culturing in a saturated humidity incubator for 24 hours, observe the adhesion to the wall, add 2-3 ml of culture medium after adhesion, and return to the incubator to continue culturing; On the 5th to 7th day after culture, the medium was completely replaced, and then half the medium was replaced every 3 days until the cells crawled out from the tissue block. After the cell confluence reached more than 70%, the cells were digested and passaged with digestion solution to obtain umbilical cord mesenchymal stem cells, which were recorded as P0 generation cells. The obtained umbilical cord mesenchymal stem cells were tested for at least one of the following items: cell activity, chromosome karyotype analysis, surface markers, and genetic disease detection; Step (8) Inoculate the P0 cells into a T25 flask at a cell density of 1E5, add 5 ml of serum-free medium, and place at 37°C and 5% CO 2 , static culture in an incubator with saturated humidity; Step (9) When the cell confluence reaches 70-80%, the cells can be subcultured. The serum-free medium supernatant is removed, 2 ml of DPBS solution is added to wash away the excess serum-free medium, which is then discarded. 1 ml of 0.25% trypsin digestion solution is taken and digested for 2 min. Two times the volume of the digestion solution is added to terminate the digestion. The cells are transferred to a centrifuge bottle tube and centrifuged at 1000 r / min for 5 min. The supernatant is discarded to obtain P1 cells.

[0026] Wherein, the DPBS solution is a DPBS buffer solution containing 1% streptomycin and penicillin.

[0027] Wherein, the disinfectant is alcohol with a volume concentration of 75%.

[0028] The length of the tissue segment is 3-4 cm; the volume of the tissue block is 0.9-1.1 mm 3 , 0.9-1.1mm3 tissue blocks, effectively preventing the formation of ice crystals in the intracellular fluid, thereby reducing damage to the cells.

[0029] The freezing solution is prepared by mixing 85% α-MEM culture medium, 5% platelet lysate, and 10% dimethyl sulfoxide in a 50 ml centrifuge tube in a volume ratio and precooling in a 4° C. refrigerator.

[0030] The serum-free medium is composed of α-MEM and platelet lysate, and specifically includes the following components (calculated by volume): inorganic salts: 200 mg / L calcium chloride, 97.67 mg / L magnesium sulfate, 400 mg / L potassium chloride, 2200 mg / L sodium bicarbonate, 6800 mg / L sodium chloride, 122 mg / L anhydrous disodium hydrogen phosphate; amino acids: 126 mg / L L-alanine-L-glutamine, 100 mg / L L-arginine hydrochloride, 31.3 mg / L L-cysteine ​​hydrochloride monohydrate, 292 mg / L L-glutamine, 42 mg / L-histidine hydrochloride monohydrate, 52 mg / L-isoleucine, 52 mg / L-leucine, 72.5 mg / L-lysine hydrochloride, 15 mg / L-methionine, 32 mg / L-phenylalanine, 48 mg / L-threonine, 51 mg / L-tyrosine disodium salt dihydrate, 1 0mg / L tryptophan, 46mg / L alanine, 50mg / L glycine; 60mg / L vitamins; 40mg / L ribonucleotides; 41mg / L deoxyribonucleotides; 1000mg / L glucose; 0.2mg / L zinc sulfate; 110mg / L sodium pyruvate; 10mg / L phenol red; 10ug / L growth factor; 1.7ug / L endothelial growth factor; 50ug / L transforming growth factor; 0.27ug / L basic fibroblast factor; 11ug / L vascular endothelial growth factor.

[0031] Wherein, the digestion solution adopts 0.25% pancreatic enzyme digestion solution. Embodiment 5

[0032] A method for establishing an umbilical cord mesenchymal stem cell seed bank, the method comprising the following steps: (1) Record the fetal information and the genetic virus detection information of the donor maternal blood in the collected umbilical cord; (2) After the obtained umbilical cord mesenchymal stem cells are passaged, cells from passages 3 to 6 are taken for cell biological property testing, stability testing, safety testing, and cell differentiation potential testing, and all relevant test data of the cells are retained; (3) After all the data are qualified, an umbilical cord mesenchymal stem cell seed bank is established. The cells are stored in liquid nitrogen at -196°C and revived and expanded when needed for use in cell therapy.

[0033] Experimental part:

[0034] 1. The umbilical cord mesenchymal stem cells obtained in Example 4 were tested for cell appearance, cell activity and cell contamination. The results showed that the cells were in good growth state, normal morphology and no contamination, which was significantly different from those cultured with serum. Figure 1-3 .

[0035] 2. Flow cytometry was used to identify surface markers of the umbilical cord mesenchymal stem cells obtained in Example 4 and the P1 cells cultured from the unfrozen tissue blocks. The cells were able to highly express CD73 and CD90 positive markers, and did not express CD34 and CD45 negative markers. The results are shown in the following table:

[0036] 3. The mesenchymal stem cells obtained in Example 4 were subjected to an experiment to identify their potential for adipogenic and osteogenic differentiation. After 20 days of induction and culture, calcium nodules appeared, indicating that human umbilical cord mesenchymal stem cells had osteogenic differentiation ability; after 20 days of induction and culture, lipid droplet structures appeared, indicating that human umbilical cord mesenchymal stem cells had adipogenic differentiation ability. Figure 4 and Figure 5 According to the above identification results, it can be shown that the method for freezing, resuscitating and preparing mesenchymal stem cells using the tissue block provided by the embodiment of the present invention is simple, efficient and can maintain its original activity. There is no difference between the umbilical cord mesenchymal stem cells obtained by tissue culture after freezing and the umbilical cord mesenchymal stem cells obtained by fresh, unfrozen tissue culture.

[0037] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A freezing solution, characterized in that: The freezing solution is composed of the following components calculated by volume percentage: α-MEM medium: 80-90% Platelet lysate: 4-6% Dimethyl sulfoxide: 8-12%.

2. A method for cryopreservation, thawing and preparation of mesenchymal stem cells from umbilical cord tissue blocks, characterized in that: It includes the following steps in sequence: Step (1) Umbilical cord collection: immerse the umbilical cord in DPBS solution and transport it to the laboratory using a biosafety transport box; Step (2) cleaning the umbilical cord: disinfect the collected umbilical cord with a disinfectant, clean it with a DPBS solution, cut it into tissue segments, and wash it again with a DPBS solution until the tissue segments turn white; Step (3) separation of Wharton's jelly: use ophthalmic forceps to separate the Wharton's jelly from the tissue segment and cut it into tissue blocks, and wash it with DPBS solution; Step (4) Cryopreservation of tissue blocks: adding a cryopreservation solution to the tissue blocks, wherein the volume of the cryopreservation solution added is one-half of the volume of the tissue blocks, and dispensing the solution into cryopreservation tubes. The cryopreservation tubes are placed in a programmed cooling cryopreservation box, and are successively refrigerated at -4°C for 2 hours, frozen at -80°C overnight, and finally frozen in liquid nitrogen at -196°C for later use; Step (5) tissue block recovery: when necessary, take out the frozen tissue block in step (4) from liquid nitrogen, thaw it in a 37°C constant temperature water bath, put it into a 15 ml centrifuge tube, add 5 times the volume of serum-free culture medium, mix and centrifuge, and remove the supernatant; Step (6) using a pipette to transfer the tissue block revived in step (5) into a culture bottle containing serum-free medium, and culturing in an incubator at 37° C., 5% CO 2 , and saturated humidity; Step (7) On the 5th to 7th day after culture, the medium is fully replaced, and then half the medium is replaced every 3 days until the cells crawl out from the tissue block. After the cell confluence reaches more than 70%, digestion solution is used for digestion and passage, thereby obtaining umbilical cord mesenchymal stem cells, which are recorded as P0 cells.

3. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue according to claim 2, characterized in that: The DPBS solution is a DPBS buffer solution containing 1% streptomycin and penicillin.

4. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: The disinfectant is alcohol with a volume concentration of 75%.

5. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: The length of the tissue segment is 3-4 cm long; the volume of the tissue block is 0.9-1.1 mm 3 .

6. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: The freezing solution is prepared by mixing 85% α-MEM culture medium, 5% platelet lysate, and 10% dimethyl sulfoxide in a 50 ml centrifuge tube in a volume ratio and precooling in a 4° C. refrigerator.

7. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: The serum-free culture medium consists of α-MEM and platelet lysate.

8. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: The digestion solution adopts pancreatic enzyme digestion solution.

9. The method for cryopreservation, thawing and preparing mesenchymal stem cells of umbilical cord tissue blocks according to claim 2, characterized in that: It also includes: Step (8) inoculating the P0 cells at a cell density of 1E5 in a T25 flask, adding 5 ml of serum-free culture medium, and placing the flask in an incubator at 37°C, 5% CO2, and saturated humidity for static culture; Step (9) When the cell confluence reaches 70-80%, the cells can be subcultured. The serum-free medium supernatant is removed, 2 ml of DPBS solution is added to wash away the excess serum-free medium, which is then discarded. 1 ml of 0.25% trypsin digestion solution is taken and digested for 2 min. Two times the volume of the digestion solution is added to terminate the digestion. The cells are transferred to a centrifuge bottle tube and centrifuged. The supernatant is discarded to obtain P1 cells.

10. A method for establishing a seed bank using the mesenchymal stem cells according to claim 2, characterized in that: The method comprises the following steps: (1) Record the fetal information and the genetic virus detection information of the donor maternal blood in the collected umbilical cord; (2) After the obtained umbilical cord mesenchymal stem cells are passaged, cells from passages 3 to 6 are taken for cell biological property testing, stability testing, safety testing, and cell differentiation potential testing, and all relevant test data of the cells are retained; (3) After all the data are qualified, an umbilical cord mesenchymal stem cell seed bank is established. The cells are stored in liquid nitrogen at -196°C and revived and expanded when needed for use in cell therapy.

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