阅读说明：本技术 改性海藻酸钠水凝胶及其制备的细胞培养基质材料和应用 (Modified sodium alginate hydrogel, cell culture matrix material prepared from same and application of cell culture matrix material ) 是由 不公告发明人 于 2018-05-29 设计创作，主要内容包括：本发明公开了一种改性海藻酸钠水凝胶,由可结合细胞因子的、且链霉亲和素修饰的纤连蛋白改性生物素偶联的海藻酸钠水凝胶制得；本发明还提供了由其制备的细胞培养基质材料及其制备的细胞培养器,含可溶性胶原蛋白、乳酸、维生素A和2-磷酸-L-抗坏血酸在内的无血清培养添加物,以及在低氧浓度条件下用于培养干细胞的方法。模拟正常干细胞在机体内生长环境,从原代培养开始获得人干细胞,具有更好的增殖活力、生物学特性和体内存活能力。这些改性海藻酸钠水凝胶的细胞培养基质材料、及其细胞培养器和无血清培养添加物,在低氧浓度条件下用于人干细胞原代和传代培养、干细胞的生物制剂,以及治疗疾病的药物组合物。(The invention discloses a modified sodium alginate hydrogel which is prepared from a streptavidin-modified fibronectin-modified biotin-coupled sodium alginate hydrogel which can be combined with cytokines; the invention also provides a cell culture substrate material prepared from the cell culture substrate material, a cell culture device prepared from the cell culture substrate material, a serum-free culture additive containing soluble collagen, lactic acid, vitamin A and 2-phospho-L-ascorbic acid, and a method for culturing stem cells under the condition of low oxygen concentration. The growth environment of normal stem cells in an organism is simulated, the human stem cells are obtained from primary culture, and the human stem cells have better proliferation activity, biological characteristics and in-vivo survival ability. The cell culture substrate material of the modified sodium alginate hydrogel, and the cell culture device and the serum-free culture additive thereof are used for primary and subculture of human stem cells, biological preparations of the stem cells under the condition of low oxygen concentration, and pharmaceutical compositions for treating diseases.)

1. A modified sodium alginate hydrogel is characterized in that the modified sodium alginate hydrogel is prepared from a streptavidin-modified fibronectin-modified biotin-coupled sodium alginate hydrogel which can be combined with cytokines.

2. The modified sodium alginate hydrogel according to claim 1, wherein the cytokine-binding streptavidin-modified fibronectin is prepared by recombining a fibronectin gene, recombining a type III domain variable region at the C-terminal thereof into a single-chain antibody capable of binding a cytokine, expressing the antibody in an Escherichia coli host to obtain fibronectin capable of binding a cytokine, and modifying the streptavidin; cytokines can bind to fibronectin; preferably, the affinity constant can reach a Kaff value of 1X 107 or more.

3. The modified sodium alginate hydrogel according to claim 1, wherein the preparation method comprises the following steps:

Firstly, performing a cross-linking reaction on biotin-coupled sodium alginate and water-soluble chitosan to obtain biotinylated sodium alginate hydrogel;

Secondly, taking the N-terminal structural domain of the cell factor as an immune antigen, and preferably selecting the molecular weight of the cell factor to be 2 kDa; immune spleen cells subjected to titer detection after inoculation of a mouse are fused with myeloma cells, and monoclonal antibodies and hybridoma cells thereof with high titer and specificity are obtained by screening after culture; designing primers for amplifying genes of variable regions of heavy chains and light chains, obtaining Single-chain antibody (scFv) gene fragments through a DNA polymerase chain reaction, cloning the scFv gene fragments to a variable region at the 3' end of a fibronectin gene through the DNA polymerase chain reaction, and then inducing and expressing fusion protein containing mouse anti-human cytokine scFv and fibronectin;

Step three, performing streptavidin labeling on fibronectin containing the mouse anti-human cytokine scFv; preferably, fibronectin containing mouse anti-human cytokine scFv is added into the biotinylated sodium alginate hydrogel, and the modified sodium alginate hydrogel is prepared by specifically binding biotin and streptavidin and binding the fibronectin containing mouse anti-human cytokine scFv to the biotinylated sodium alginate hydrogel.

4. The modified sodium alginate hydrogel according to any one of claims 1 to 3, wherein fibronectin containing mouse anti-human cytokine scFv is bound to the sodium alginate hydrogel by specific binding of biotin to streptavidin; preferably, coupling sodium alginate by biotin, and then performing crosslinking reaction with water-soluble chitosan and glutaraldehyde to obtain sodium alginate hydrogel; more preferably, the sodium alginate hydrogel is in a porous structure, and the hardness is 0.2-50 kPa.

5. A method of preparing a modified sodium alginate hydrogel as claimed in any one of claims 1 to 4.

6. A preparation method of the modified sodium alginate hydrogel is characterized by comprising the following steps:

firstly, labeling biotin on sodium alginate through carboxyl according to a certain proportion, and performing crosslinking reaction with water-soluble chitosan and glutaraldehyde to obtain sodium alginate hydrogel;

Secondly, taking the N-end structural domain of the cell factor as an immune antigen, inoculating the immune spleen cell subjected to titer detection to a mouse, fusing with a myeloma cell, and screening after culture to obtain a monoclonal antibody with high titer and specificity and a hybridoma thereof; obtaining scFv gene segment of anti-human cell factor by DNA polymerase chain reaction;

Thirdly, cloning the scFv gene fragment to the variable region at the 3' end of the fibronectin gene by a PCR technology, and then inducing and expressing the fusion protein containing the mouse anti-human cytokine scFv and fibronectin; labeling fibronectin containing mouse anti-human cytokine scFv with streptavidin to obtain streptavidin-labeled fibronectin containing mouse anti-human cytokine scFv;

and fourthly, combining fibronectin containing mouse anti-human cytokine scFv to the sodium alginate hydrogel through specific binding of biotin and streptavidin to prepare the modified sodium alginate hydrogel.

7. A modified sodium alginate hydrogel prepared according to the method of claim 6.

8. A cell culture matrix material, comprising the modified sodium alginate hydrogel according to any one of claims 1 to 7.

9. The cell culture matrix material according to claim 8, wherein the cell culture matrix material is prepared by adding a cytokine to the modified sodium alginate hydrogel according to any one of claims 1 to 7, wherein the cytokine can specifically bind to the modified sodium alginate hydrogel; preferably, the cytokine binds specifically to the murine anti-human cytokine scFv on fibronectin in the modified sodium alginate hydrogel.

10. A cell culture matrix material according to any one of claims 8-9, wherein the cytokines that bind to fibronectin include epidermal growth factor, basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte factor, platelet derived growth factor, insulin-like growth factor, nerve growth factor, keratinocyte or stem cell factor; epidermal growth factor and basic fibroblast growth factor are preferred.

11. A cell culture device comprising a cell culture matrix material according to any one of claims 8 to 10, wherein the cell culture matrix material is added to the cell culture device for plating; preferably forming a 0.5-5mm film; preparing a cell culture device containing coated cell culture matrix materials;

12. A serum-free culture supplement for stem cell culture, wherein the serum-free culture supplement comprises soluble collagen, lactic acid, vitamin A and 2-phospho-L-ascorbic acid, and optionally recombinant human serum albumin, recombinant human insulin, recombinant human transferrin, glucose, sodium bicarbonate and/or buffer HEPES.

13. the serum-free culture supplement of claim 12, wherein the serum-free culture supplement is used at a concentration of 1-fold, i.e., 0.5-1000ng/mL of soluble collagen, 0.1-200mmol/L of lactic acid, 0.1-500 μmol/L of vitamin a, 0.5-500ng/mL of 2-phospho-L-ascorbic acid, and 0.5-200 μ g/mL of recombinant human serum albumin, 0.5-200 μ g/mL of recombinant human insulin, 0.1-500ng/mL of recombinant human transferrin, 0.1-10% of glucose, 3mM of sodium bicarbonate, and 5mM of HEPES buffer; the percentage of glucose is mass volume percentage, and the unit of glucose is g/mL.

14. The serum-free culture supplement of any one of claims 12-13, wherein the serum-free culture supplement has a preservation concentration or lyophilized powder of 10-200 fold.

15. A method for culturing stem cells under low oxygen concentration conditions, which comprises culturing stem cells under low oxygen concentration culture conditions using the modified sodium alginate hydrogel according to any one of claims 1 to 7 or the cell culture matrix material according to any one of claims 8 to 10, or the cell culture device according to claim 11 and the serum-free culture supplement according to any one of claims 12 to 14.

16. use of a modified sodium alginate hydrogel as defined in any one of claims 1 to 7 or a cell culture matrix material as defined in any one of claims 8 to 10 or a cell culture device as defined in claim 11 or a serum-free culture supplement as defined in any one of claims 12 to 14 in a pharmaceutical composition.

Technical Field

the invention relates to a modified sodium alginate hydrogel which is prepared from a streptavidin-modified fibronectin-modified biotin-coupled sodium alginate hydrogel which can be combined with cytokines; a cell culture substrate material prepared by the same, a cell culture device prepared by the same and a method for culturing stem cells under the condition of low oxygen concentration by using serum-free culture additives containing soluble collagen, lactic acid, vitamin A and 2-phosphoric acid-L-ascorbic acid. In particular, the human stem cells are obtained from primary culture by simulating the growth environment of normal stem cells in an organism, and have better proliferation activity, biological characteristics and in-vivo survival ability.

background

at present, most of the culture methods for stem cells in scientific research and production are simple plastic culture bottles or culture dishes or culture plates, and culture media containing cytokines are added for culture. Most of stem cells grow adherently, the hard plastic bottle bottom is not favorable for the growth of the stem cells, and the biological functions of the stem cells obtained by culture are greatly changed. Therefore, attempts have been made to add various substrates which are beneficial to the growth of stem cells, such as Matrigel, Geltrex Matrigel, fibronectin, soluble collagen, sodium alginate, chitosan, etc., to the bottom of a plastic culture bottle or a culture dish or a culture plate, or to better exhibit the biological functions of the stem cells on microspheres by utilizing the existing pore size and hardness for three-dimensional culture of the stem cells.

Researchers also embed the stem cells in the gel directly and then culture the stem cells in vitro, and although the culture mode increases the proliferation of the stem cells and inhibits the apoptosis of the cells, the expansion and subculture of the stem cells add a lot of troubles; is not favorable for obtaining stem cells.

The three-dimensional environment obtained by the method is far from the three-dimensional growth mode of the stem cells of the extracellular matrix in the real body. Besides the reticular scaffold, the extracellular matrix also needs various proteins and cytokines adhered to the reticular scaffold, growth environment and other factors to jointly create the optimal growth condition for the stem cells. Through the growth conditions, the proliferation capacity, the anti-apoptosis capacity, the in vivo and in vitro survival capacity and the biological function of the stem cells reach better states, which is beneficial to the in vivo better tissue organ repair, the improvement of the microenvironment of the organism and the capability of secreting cell factors, and the capability of treating diseases in a true sense is achieved.

Disclosure of Invention

The invention relates to a modified sodium alginate hydrogel which is prepared from a sodium alginate hydrogel which can be combined with a cytokine and is coupled with fibronectin modified biotin and can be combined with a cytokine, wherein the modified sodium alginate hydrogel is prepared by research and development teams for years and simulates the growth condition of normal stem cells in vivo, including factors such as extracellular matrix, nutrient components, oxygen concentration and the like; a cell culture substrate material prepared by the same, a cell culture device prepared by the same and a method for culturing stem cells under the condition of low oxygen concentration by using a serum-free culture additive containing soluble collagen, lactic acid, vitamin A and 2-phosphoric acid-L-ascorbic acid. Sodium alginate is used as a bracket, and biotin is coupled to prepare active biotin-sodium alginate hydrogel; biotin specifically binds to streptavidin, which modifies fibronectin, i.e. forms the basic framework of the extracellular matrix.

Since each subunit of fibronectin has six functional regions and an RGD sequence, fibronectin can bind to various substances to exert a strong function. Fibronectin, by adhering to cells and nutrients, transports nutrients (such as soluble collagen, hyaluronic acid, small peptides, glycoproteins, etc.) required for cellular metabolism, growth, proliferation, and regulates the extracellular microenvironment. In particular, fibronectin has domains similar to those of antibodies, and variable regions exist in the type III domain, which can be inserted into the complementarity determining regions of monoclonal antibodies, and which can react with and specifically bind to the target protein to produce antigen-antibody reaction. By utilizing the characteristic, the single-chain antibody of the mouse anti-human cytokine is inserted into the fibronectin type III domain through gene recombination, so that the cytokine can be specifically combined. Meanwhile, the areas of the I type and II type fibronectin structure domains which are specifically combined with soluble collagen are utilized, and the soluble collagen is added to be specifically combined with fibronectin to form a cell growth environment similar to extracellular matrix.

In addition to further simulating the in vivo cell growth conditions, we found that the oxygen concentration in the tissues and organs in vivo is substantially between 1-10%, and the cell growth conditions are more suitable and viable for a longer time under the oxygen concentration conditions. Through our experiments, it was found that the proliferation rate and biological function of the stem cells cultured under the low oxygen condition are better than those of the stem cells cultured under the normal oxygen condition even in the ordinary culture device without any feeder layer. The proliferation rate of stem cells cultured in the culture device of sodium alginate gel combined with fibronectin containing cytokines is further superior to that of stem cells cultured in a common culture device. Particularly, the stem cells better maintain the proliferation capacity and the stem cell characteristics and the in vivo survival capacity under the culture condition containing soluble collagen, lactic acid, vitamin A and 2-phospho-L-ascorbic acid. The research shows that the soluble collagen can be combined with fibronectin to form an extracellular matrix microenvironment which is beneficial to the growth of stem cells; lactic acid and vitamin A help stem cells to maintain better proliferation capacity under an anoxic condition and are substrates for generating effective energy of the stem cells, vitamin A and 2-phospho-L-ascorbic acid better maintain the dryness characteristics of the stem cells, namely self-renewal and multi-directional differentiation potential, and the molecules jointly form a microenvironment for stem cell growth and synergistically ensure the proliferation growth and the functional exertion of the stem cells, so that a novel serum-free culture additive for the stem cells is discovered on the basis of the research.

The invention provides a modified sodium alginate hydrogel which is characterized by being prepared from a streptavidin-modified fibronectin-modified biotin-coupled sodium alginate hydrogel which can be combined with cytokines.

Preferably, the sodium alginate hydrogel is biotin-modified sodium alginate, namely, the sodium alginate contains carboxyl, and biotin molecules are marked on the sodium alginate hydrogel according to the molar ratio of 1: 0.1-0.5; and then the sodium alginate hydrogel is subjected to crosslinking reaction with water-soluble chitosan and glutaraldehyde to obtain the sodium alginate hydrogel which is in a porous structure and has the hardness of 0.2-50 kPa.

Preferably, the cytokine-binding streptavidin-modified fibronectin is prepared by mutating a type III domain variable region of fibronectin gene into a sequence of a mouse anti-human cytokine Single-chain antibody (Single chain Fv, scFv), expressing the fibronectin in an escherichia coli host to obtain fibronectin capable of binding the cytokine, modifying streptavidin, and adding the cytokine to bind to fibronectin; more preferably, the affinity constant can reach a Kaff value of 1 × 107 or more.

Preferably, the obtained mouse anti-human cytokine single-chain antibody takes a cytokine N-terminal structural domain as an immune antigen; more preferably, it has a molecular weight of 2 kDa; immune spleen cells subjected to titer detection after being inoculated to mice are fused with myeloma cells, and monoclonal antibodies (mAbs) with high titer and specificity and hybridoma cells thereof are obtained by screening after culture. Primers for amplifying heavy chain and light chain variable region genes are designed, scFv gene fragments are obtained through DNA Polymerase Chain Reaction (PCR) and splicing-overlap extension (SOE) PCR, the scFv gene fragments are cloned to a variable region at the 3' end of a fibronectin gene through a PCR technology, and then fusion protein containing mouse anti-human cytokine scFv and fibronectin is induced and expressed. And performing streptavidin labeling on fibronectin containing the mouse anti-human cytokine scFv, adding the labeled streptavidin labeled fibronectin into biotinylated sodium alginate hydrogel, and specifically binding the fibronectin containing the mouse anti-human cytokine scFv to the sodium alginate hydrogel through biotin and streptavidin.

the invention also provides a method for preparing the modified sodium alginate hydrogel, which is characterized by comprising the following steps:

Firstly, labeling biotin on sodium alginate by carboxyl in proportion, and performing crosslinking reaction on glutaraldehyde and water-soluble chitosan to obtain sodium alginate hydrogel;

Secondly, taking the N-end structural domain of the cell factor as an immune antigen, inoculating the immune spleen cell subjected to titer detection to a mouse, fusing with a myeloma cell, and screening after culture to obtain a monoclonal antibody with high titer and specificity and a hybridoma thereof; obtaining scFv gene segment of anti-human cell factor by DNA PCR;

thirdly, cloning the scFv gene fragment to the variable region at the 3' end of the fibronectin gene by a PCR technology, and then inducing and expressing the fusion protein containing the mouse anti-human cytokine scFv and fibronectin; labeling fibronectin containing mouse anti-human cytokine scFv with streptavidin to obtain streptavidin-labeled fibronectin containing mouse anti-human cytokine scFv;

and fourthly, combining fibronectin of the mouse anti-human cytokine scFv to the sodium alginate hydrogel through specific binding of biotin and streptavidin to prepare the modified sodium alginate hydrogel.

optionally, the cell culture matrix material of the sodium alginate hydrogel combined with the fibronectin containing the cell factor is prepared by adding the cell factor into the fibronectin of the sodium alginate hydrogel combined with the mouse anti-human cytokine scFv, and combining the mouse anti-human cytokine scFv specifically with the fibronectin.

Preferably, the sodium alginate-modified biotin provided by the invention has specific binding with fibronectin-modified streptavidin, and forms a good extracellular matrix in a culture device scaffold containing cytokines and fibronectin.

The invention also provides a cell culture matrix material, wherein the cell factor is added into the modified sodium alginate hydrogel, and the cell factor can be specifically combined with the modified sodium alginate hydrogel, so that the cell culture matrix material is prepared; preferably, the cytokine binds specifically to the murine anti-human cytokine scFv on fibronectin in the modified sodium alginate hydrogel.

Preferably, the cytokines capable of binding to recombinant fibronectin include Epidermal Growth Factor (EGF), basic fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF), Hepatocyte Growth Factor (HGF), Platelet Derived Growth Factor (PDGF), insulin like Growth Factor (insulin like Growth Factor, IGF), Nerve Growth Factor (NGF), Keratinocyte Growth Factor (KGF), and Stem cell Factor (Stem cell Growth Factor, SCF), etc.; preferably, the cytokines commonly used in stem cell culture, namely EGF and bFGF.

The invention also provides a cell culture device, wherein the cell culture device is used for adding the cell culture substrate material into the cell culture device for bottom paving; preferably, a 0.5-5mm film is continuously and uniformly formed on the surface of the cell culture device; preparing a cell culture device containing coated cell culture matrix materials;

The invention also provides a serum-free culture additive for stem cell culture, wherein the serum-free culture additive comprises soluble collagen, lactic acid, vitamin A, 2-phosphoric acid-L-ascorbic acid, recombinant human albumin, recombinant human insulin, recombinant human transferrin, glucose, sodium bicarbonate and buffer HEPES.

The serum-free culture additive has the use concentration of 1 time, namely 0.5-1000ng/mL of soluble collagen, 0.1-200mmol/L of lactic acid, 0.1-500 mu mol/L of vitamin A, 0.5-500ng/mL of 2-phosphoric acid-L-ascorbic acid, 0.5-200 mu g/mL of recombinant human serum albumin, 0.5-200 mu g/mL of recombinant human insulin, 0.1-500ng/mL of recombinant human transferrin, 0.1-10% (mass-volume ratio, g/mL) of glucose, 3mM of sodium bicarbonate and 5mM of HEPES buffer solution. The serum-free culture medium at a 1-fold concentration is the final concentration for stem cell culture, and is preferably at a 10-200-fold storage concentration for better storage and addition to the culture medium. High concentration storage is more advantageous to maintain the activity of its ingredients.

Preferably, the serum-free culture additive solution is placed in a freeze dryer for freeze drying to obtain the serum-free culture additive freeze-dried powder, and the serum-free culture additive freeze-dried powder is sealed by a gland and then placed in a refrigerator at the temperature of 2-8 ℃ for storage, transportation and preservation, so that the storage and transportation are facilitated, and the effective period is longer.

The serum-free culture additive contains soluble collagen, active protein and saccharide, and can be specifically combined with the I-type and II-type fibronectin domains to form a complete extracellular matrix environment, which is more favorable for stem cell growth and biological function maintenance.

The invention also provides a method for co-culturing stem cells under the condition of low oxygen concentration by combining the cell culture matrix material with the serum-free culture additive, wherein the method comprises the steps of separating and purifying single-cell suspension from human tissues, and filtering the single-cell suspension by using a 40-micrometer cell sieve; centrifuging the obtained cell suspension at 1000 rpm for 10min, washing the cell precipitate with 1 × PBS (pH7.4) for 1 time, adding cell culture solution containing 1 time of serum-free culture additive, re-suspending, and adding 1-10 × 106 cells/mL into a cell culture container coated with the cell culture matrix material after staining and counting; or cutting the tissue to about 1mm3 by ophthalmic scissors, resuspending with 1 XPBS (pH7.4), centrifuging at 1000 rpm for 10 minutes, discarding the supernatant, resuspending the tissue pellet with 1 time of cell culture solution containing serum-free culture additive, adding into a cell culture device coated with the cell culture matrix material, and growing at 37 deg.C in a three-gas culture box with 5% CO2 and low oxygen concentration of 1-10% for 48 hours;

After culturing for 48 hours, changing the culture solution of the stem cells, sucking the culture solution by using a 5mL pipette, adding a cell culture solution containing 1 time of serum-free culture additives, continuously placing the cells in a cell culture device coated with the cell culture substrate material in a three-gas culture box with 37 ℃, 5% CO2 and low oxygen concentration of 1-10% for continuous culture, and changing the culture solution for 1 time by using a fresh culture solution at intervals of 2 days;

Growing and fusing the stem cells to more than 80% in a cell culture device, sucking and removing culture solution by using a 5mL pipette, washing the bottom of a primary culture bottle by taking 1 XPBS (pH7.4), adding pancreatin (containing EDTA (0.02 percent) in a mass-volume ratio) of 0.25 percent into the culture bottle, placing the culture bottle at 37 ℃, and digesting the cells in a 5 percent CO2 incubator for 2 to 3 minutes; after digestion, adding fetal calf serum to stop digestion, adding physiological saline, blowing and beating by using a 5mL pipette, sucking into a 15mL centrifuge tube, washing once by using the physiological saline, adding into the same 15mL centrifuge tube, centrifuging at 1000 rpm for 5 minutes, and collecting stem cells; after centrifugation, the suspension is resuspended by fresh culture solution, counted, fresh cell culture solution containing 1 time of serum-free culture additives is added by a 5mL pipette, the suspension is added into 1 new cell culture device for coating the cell culture matrix material to continue to grow in a three-gas incubator with 37 ℃, 5% CO2 and low oxygen concentration of 1-10%, and the fresh cell culture solution containing 1 time of serum-free culture additives is replaced every 2 days; when the stem cells grow and fuse to more than 80 percent, collecting the stem cells by trypsinization for continuous passage for 10 generations for culture to obtain stem cells with the magnitude of 1011 magnitude or more, resuspending the stem cells by using normal saline, dyeing and counting the orchis ensiformis to obtain the stem cells, and storing the stem cells in a refrigerator at 4 ℃ for later use;

And storing part of the stem cells in a freezing storage solution consisting of 40% of stem cell culture solution, 10% of dimethyl sulfoxide and 50% of fetal calf serum in a liquid nitrogen tank at the temperature of-196 ℃ for later recovery.

The human tissue comprises waste umbilical cord, cord blood, placenta, amnion, fat, bone marrow, skin, menstrual blood, teeth, synovial fluid, synovial membrane, endometrium, brain tissue, liver tissue, pancreas, retina, etc., culturing in a culture apparatus with corresponding hardness of sodium alginate hydrogel matrix to obtain corresponding stem cells, namely umbilical cord Mesenchymal Stem Cells (MSC), umbilical cord blood Mesenchymal stem cells, placental sub-totipotent stem cells, amniotic membrane stem cells, adipose stem cells, bone marrow Mesenchymal stem cells, bone marrow Endothelial Progenitor Cells (EPCs), epidermal stem cells, dermal fibroblast stem cells, Menstrual blood endometrial progenitor cells (MenSC), dental pulp stem cells, synovial fluid Mesenchymal stem cells, synovial membrane Mesenchymal stem cells, endometrial stem cells, neural stem cells, hepatic stem cells, pancreatic stem cells, retinal progenitor cells, and the like.

the hypoxic culture condition is that the hypoxic concentration is 1-10%; preferably, a low oxygen concentration of 1-5% is selected.

Compared with the 10 th generation stem cell cultured under the condition of low oxygen concentration, the stem cell prepared after 30 generations of subculture has stronger cell proliferation rate and higher clone forming rate, also maintains good cell morphology and cell phenotype of the stem cell, and can differentiate corresponding adult cells; preferably, the prepared stem cells have a longer survival time in animals and have good stem cell characteristics.

The stem cells cultured under the low-oxygen concentration culture condition comprise the cell culture matrix material and the cell culture solution of the serum-free culture additive, and the stem cells cultured under the low-oxygen concentration culture condition can be prepared into biological products and pharmaceutical compositions for treating diseases. In particular to a cell therapy product prepared from nervous system diseases, cardiovascular and cerebrovascular diseases, orthopedic diseases, autoimmune diseases, diabetes, nephropathy, medical plastic and the like, and is used for clinical treatment.

Drawings

FIG. 1 is a surface topography of a cell culture device scanning electron microscope of the biotin-modified sodium alginate hydrogel prepared in example 1

FIG. 2 is a Western blot of a fibronectin fusion protein of anti-human EGF and bFGF single-chain antibody prepared in example 3

FIG. 3 is a schematic diagram of a cell culture apparatus with biotin-modified sodium alginate hydrogel coupled to cytokine-containing fibronectin

FIG. 4 is a graph of mesenchymal stem cells of generations 10, 20 and 30 cultured under low oxygen concentration conditions in a cell culture apparatus in which sodium alginate hydrogel prepared in example 5 binds fibronectin containing EGF and bFGF

FIG. 5 is a graph of 10 th, 20 th and 30 th generation endothelial progenitor cells cultured under low oxygen concentration conditions in a cell culture apparatus in which the sodium alginate hydrogel prepared in example 5 binds fibronectin containing VEGF, IGF-1 and EGF

FIG. 6 is a graph of the 10 th, 20 th and 30 th generation endometrial stem cells cultured under the low oxygen concentration condition in the cell culture device of the sodium alginate hydrogel combined with fibronectin containing insulin and bFGF prepared in example 5

FIG. 7 is a graph of cell proliferation curves comparing the 30 th generation MSCs prepared in example 7 with the 10 th generation MSCs prepared in the control group

FIG. 8 is a graph of cell proliferation curves comparing the 30 th generation EPC prepared in example 8 with the 10 th generation EPC prepared in the control

FIG. 9 is a graph showing cell proliferation profiles of MenSC generation 30 prepared in example 9 compared with MenSC generation 10 prepared in control group

FIG. 10 is a diagram showing the flow detection result of the 30 th generation mesenchymal stem cell prepared in example 7

FIG. 11 is ALP staining pattern (A) of the 3-week-old cells induced by osteogenesis of MSC of generation 30 prepared in example 7, reticulocyte formation of EPC of generation 30 prepared in example 8 (B), and Asin blue positive pattern (C) of differentiation of MenSC of generation 30 prepared in example 9 into cartilage observed under an inverted microscope

FIG. 12 is a graph of the change in area of the cavity at the 4 th week after transplantation of P30-MSC prepared in example 7 and control P10-MSC

FIG. 13 is a green fluorescence pattern observed under an inverted fluorescence microscope at 4 weeks after transplantation of P30-MSC prepared in example 7 and control P10-MSC

Detailed Description

as used herein, the term "sodium alginate" is a natural polysaccharide isolated from brown seaweeds such as kelp and gulfweed, which is an anionic copolymer, has good biocompatibility and low toxicity, and has a strong gelling ability. The sodium alginate and the chitosan can quickly generate cross-linking reaction to generate gel. By utilizing the property, the gel formed by the sodium alginate and the chitosan has thermal irreversibility, the gel performance is not influenced by temperature, and the gel can be subjected to heating sterilization, microwave oven and other treatments.

sodium alginate as a linear polymer is formed by connecting three chain segments through glycosidic bonds, each structural unit of the molecule has two secondary hydroxyl groups, and the secondary hydroxyl groups have the reactivity of alcoholic hydroxyl groups. In the invention, biotin is marked by carboxyl according to the molar ratio of 1: 0.1-0.5, and the biotin and water-soluble chitosan can generate cross-linking reaction under the action of glutaraldehyde to form hydrogel.

the sodium alginate hydrogel is biotin-modified sodium alginate, is obtained by performing crosslinking reaction with water-soluble chitosan and glutaraldehyde, has a porous structure and hardness of 0.2-50kPa, and continuously and uniformly forms a 0.5-5mm thin film on the surface of a cell culture device. The prepared sodium alginate hydrogel cell culture device can be used for culturing different stem cells suitable for growing under a specific hardness, such as stem cells suitable for culturing fat, liver, lung, embryo, fat and nerve parts under a hardness of 0.1-1 kPa; the stem cells of muscle and heart parts are cultured under the hardness of 2-12 kPa.

as used herein, the term "fibronectin" is a high molecular weight glycoprotein having a subunit relative molecular mass of 22-25 ten thousand. Each subunit forms disulfide bonds at the C-terminus to form a dimer or multimer, i.e., each subunit of fibronectin is composed of several domains, and has six functional regions such as binding sites with high affinity for cell surface receptors, collagen, fibrin, and sulfated proteoglycans, in which the RGD tripeptide sequence is found to be the smallest structural unit recognized by cells.

In particular, fibronectin has domains similar to those of antibodies, and variable regions exist in the type III domain, which can be inserted into antibody complementarity determining regions, and which can react with and specifically bind to a target protein to produce an antigen-antibody reaction. By utilizing the characteristic, the gene recombination is carried out in the fibronectin type III domain to insert the mouse anti-human cytokine scFv, so that the specific binding of the cytokine can be realized. Meanwhile, the areas of the I type and II type fibronectin structure domains which are specifically combined with soluble collagen are utilized, and the soluble collagen is added to be specifically combined with fibronectin to form a cell growth environment similar to extracellular matrix.

the streptavidin modified fibronectin capable of binding the cytokine protein is obtained by recombining fibronectin gene, inserting a sequence of mouse anti-human cytokine scFv into a type III domain variable region gene, expressing the recombinant fibronectin in an escherichia coli host, obtaining the fibronectin capable of binding the cytokine protein, modifying the streptavidin, adding the cytokine to bind to the fibronectin, and the affinity constant can reach a Kaff value of more than 5 multiplied by 107.

A cell factor N-terminal structural domain is used as an immune antigen, a Balb/c mouse is subjected to intraperitoneal injection at multiple points after emulsification of Freund complete adjuvant and antigen, after 2 weeks, blood is taken after tail breaking to measure antibody titer, enzyme linked immunosorbent assay (ELISA) serum antibody titer detection is selected, a Balb/c mouse with ELISA serum antibody titer detection reaching more than 1: 104 is selected for fusion, before 3 days of fusion, the antigen is subjected to intraperitoneal injection for 1 time without adjuvant, 50 mu g of the antigen is obtained, and then separation and purification are carried out to obtain immune splenocytes. Taking normal Balb/c mouse spleen cells as feeder cells (2 multiplied by 105/hole), taking myeloma cell strains SP 2/01 multiplied by 107 cells in logarithmic phase and 1 multiplied by 108 immune spleen cells, fusing with 500mL/L PEG according to a conventional method, detecting culture supernatant by using an indirect ELISA method, selecting strong positive clones, purifying antibodies and identifying antibody specificity and affinity to obtain specific monoclonal antibodies (mAb) and hybridoma cells thereof. This technical scheme is well known to those skilled in the art.

According to the sequences of a mouse antibody V region and a CHl region, primers are designed according to the mouse antibody family such as Orland and the like, 1 pair of each of a heavy Chain variable region primer and a light Chain variable region primer is respectively designed and amplified according to different subclasses of VH, total RNA of the obtained specific hybridoma cells is extracted and screened, complementary DNA is subjected to reverse transcription, and then the heavy Chain variable region (VH) and the light Chain variable region (VL) gene segments are respectively obtained through amplification by Polymerase Chain Reaction (PCR). Meanwhile, primers can be designed to recombine the variable region PCR technology of the heavy chain and the light chain together to form a single-chain antibody gene fragment (VH-Linker-VL). The obtained variable region or single-chain antibody gene fragment is recombined to the fibronectin type III structural domain variable region by a PCR technology, then cloned to a plasmid, and after the gene sequence is verified to be correct by sequencing, recombinant protein expression is carried out in escherichia coli. And obtaining fusion protein of fibronectin and mouse anti-human cytokine scFv after protein purification, and performing streptavidin modification by using a streptavidin labeling kit to finally obtain the streptavidin labeled fibronectin containing the mouse anti-human cytokine scFv.

the streptavidin-labeled fibronectin containing the mouse anti-human cytokine scFv is added into the biotin-modified sodium alginate hydrogel in a molar ratio of 1: 1-5, and the biotin and the streptavidin are utilized to have specific binding, so that the surface of the sodium alginate gel is specifically bound with the fibronectin containing the mouse anti-human cytokine scFv.

The invention also provides a method for preparing the modified sodium alginate hydrogel, which is characterized by comprising the following steps:

Firstly, labeling biotin on sodium alginate by carboxyl in proportion, and performing crosslinking reaction on glutaraldehyde and water-soluble chitosan to obtain sodium alginate hydrogel;

Secondly, taking the N-end structural domain of the cell factor as an immune antigen, inoculating the immune spleen cell subjected to titer detection to a mouse, fusing with a myeloma cell, and screening after culture to obtain a monoclonal antibody with high titer and specificity and a hybridoma thereof; obtaining scFv gene segment of anti-human cell factor by DNA polymerase chain reaction;

Thirdly, cloning the scFv gene fragment to the variable region at the 3' end of the fibronectin gene by a PCR technology, and then inducing and expressing the fibronectin fusion protein containing the mouse anti-human cytokine scFv; labeling fibronectin containing mouse anti-human cytokine scFv with streptavidin to obtain streptavidin-labeled fibronectin containing mouse anti-human cytokine scFv;

And fourthly, combining fibronectin of the mouse anti-human cytokine scFv to the sodium alginate hydrogel through specific binding of biotin and streptavidin to prepare the modified sodium alginate hydrogel.

As used herein, the term "cell culture substrate" is a cell growth material prepared from natural high molecular materials such as chitosan, silk fibroin, collagen, and the like, has weak foreign body antigenicity, good biocompatibility, is biodegradable, and can promote cell adhesion, growth, proliferation, and the like. Optionally, when a cytokine is added in an equimolar amount to fibronectin containing a mouse anti-human cytokine scFv, after binding overnight at 4 ℃ in a refrigerator, the solution is discarded and sufficiently dried in a sterile environment, that is, fibronectin containing an anti-cytokine scFv specifically binds the cytokine, to prepare a cell culture matrix material in which a sodium alginate hydrogel binds fibronectin containing the cytokine.

The cytokines capable of binding to recombinant fibronectin include epidermal growth factor, basic fibroblast growth factor, vascular endothelial growth factor, hepatocyte factor, platelet-derived growth factor, insulin-like growth factor, nerve growth factor, keratinocyte factor, stem cell factor and the like, and preferably, cytokines commonly used in stem cell culture, i.e., epidermal growth factor and basic fibroblast growth factor.

As used herein, the term "cell culture device" is a device made of polymer material, such as polystyrene, polyvinyl chloride, and polyethylene. The invention also provides a cell culture device, wherein the modified sodium alginate hydrogel is placed into the cell culture device, a 0.5-5mm thin film is continuously and uniformly formed on the surface of the cell culture device, and then cell factors are added, wherein the cell factors can be specifically combined with the modified sodium alginate hydrogel, so that the cell culture substrate material is prepared; preferably, the cytokine binds specifically to the murine anti-human cytokine scFv on fibronectin in the modified sodium alginate hydrogel.

The cell culture apparatus adopts cell culture dishes with various diameters (such as 35mm, 60mm, 90mm, 100mm, 150mm and 245mm square), cell culture bottles with various specifications (such as 25cm2, 75cm2, 150cm2, 175cm2 and 225cm 2), cell culture plates with various sizes (such as 96 wells, 24 wells, 12 wells, 6 wells and 4 wells), multi-layer cell culture bottles (such as 2 layers, 3 layers, 4 layers and 5 layers) and multi-layer cell culture apparatus (such as 1 layer type, 2 layers type, 5 layers type, 10 layers type and 40 layers type), and polystyrene materials for promoting stem cell growth are used.

The cell culture device can be stored at 2-8 deg.C, -10 deg.C, -20 deg.C, -56 deg.C and-86 deg.C after vacuum sealing, preferably at-20 deg.C for a limited period of 6 months.

As used herein, the term "serum-free culture supplement" is a nutrient that provides nutrients, such as amino acids, vitamins, lipids, cholesterol, glucose, and/or growth factors, that aid in cell growth without the addition of any animal serum. The serum-free culture additive contains soluble collagen, active protein and saccharide, and can be specifically combined with I-type and II-type fibronectin domains to form a complete extracellular matrix environment, thereby being more beneficial to the growth of stem cells and the maintenance of biological functions.

the invention provides a serum-free culture additive for stem cell culture, wherein the serum-free culture additive is 1-fold concentration and comprises the following components:

1) Soluble collagen: 0.5-1000 ng/mL;

2) Lactic acid: 0.1-200 mmol/L;

3) Vitamin A: 0.1-500 mu mol/L;

4) 2-phosphoric acid-L-ascorbic acid: 0.5-500 ng/mL;

5) Recombinant human serum albumin: 0.5-200 mug/mL;

6) recombinant human insulin: 0.5-200 mug/mL;

7) Recombinant human transferrin: 0.1-500 ng/mL;

8) Glucose: 0.1-10% (mass/volume);

9) Sodium bicarbonate: 3 mM;

10) HEPES buffer solution: 5 mM.

Preferably 10-200 times, the serum-free culture additive is filled in a glass bottle or a plastic bottle, the filling volume is 5-100mL according to different specification requirements, and the serum-free culture additive can be stored at 2-8 ℃, 10 ℃, 20 ℃, 56 ℃ and 86 ℃ after being screwed and sealed, preferably at 86 ℃ for a limited period of 12 months.

Preferably, the filled penicillin bottle or plastic bottle is placed in a freeze dryer for freeze drying to obtain the serum-free culture additive freeze-dried powder, and the serum-free culture additive freeze-dried powder is sealed by a gland and then placed in a refrigerator at the temperature of 2-8 ℃ for storage, transportation and preservation, so that the storage and transportation are facilitated, and the effective period is longer.

According to the above invention, the present invention also provides a method for culturing stem cells under the condition of low oxygen concentration by using the above cell culture substrate material, wherein the method comprises the steps of separating and purifying single cell suspension from human tissues, and filtering the single cell suspension by using a 40 μm cell sieve; centrifuging the obtained cell suspension at 1000 rpm for 10min, washing the cell precipitate with 1 × PBS (pH7.4) for 1 time, adding cell culture solution containing 1 time of serum-free culture additive, re-suspending, and adding 1-10 × 106 cells/mL into a cell culture container coated with the cell culture matrix material after staining and counting; or cutting the tissue to about 1mm3 by ophthalmic scissors, resuspending with 1 XPBS (pH7.4), centrifuging at 1000 rpm for 10 minutes, discarding the supernatant, resuspending the tissue pellet with 1 time of cell culture solution containing serum-free culture additive, adding into a cell culture device coated with the cell culture matrix material, and growing at 37 deg.C in a three-gas culture box with 5% CO2 and low oxygen concentration of 1-10% for 48 hours;

After culturing for 48 hours, changing the culture solution of the stem cells, sucking the culture solution by using a 5mL pipette, adding a cell culture solution containing 1 time of serum-free culture additives, continuously placing the cells in a cell culture device coated with the cell culture substrate material in a three-gas culture box with 37 ℃, 5% CO2 and low oxygen concentration of 1-10% for continuous culture, and changing the culture solution for 1 time by using a fresh culture solution at intervals of 2 days;

Growing and fusing the stem cells to more than 80% in a cell culture device, sucking and removing culture solution by using a 5mL pipette, washing the bottom of a primary culture bottle by taking 1 XPBS (pH7.4), adding pancreatin (containing EDTA (0.02 percent) in a mass-volume ratio) of 0.25 percent into the culture bottle, placing the culture bottle at 37 ℃, and digesting the cells in a 5 percent CO2 incubator for 2 to 3 minutes; after digestion, adding fetal calf serum to stop digestion, adding physiological saline, blowing and beating by using a 5mL pipette, sucking into a 15mL centrifuge tube, washing once by using the physiological saline, adding into the same 15mL centrifuge tube, centrifuging at 1000 rpm for 5 minutes, and collecting stem cells; after centrifugation, the suspension is resuspended by fresh culture solution, counted, fresh cell culture solution containing 1 time of serum-free culture additives is added by a 5mL pipette, the suspension is added into 1 new cell culture device for coating the cell culture matrix material to continue to grow in a three-gas incubator with 37 ℃, 5% CO2 and low oxygen concentration of 1-10%, and the fresh cell culture solution containing 1 time of serum-free culture additives is replaced every 2 days; when the stem cells grow and fuse to more than 80 percent, collecting the stem cells by trypsinization for continuous passage for 10 generations for culture to obtain stem cells with the magnitude of 1011 magnitude or more, resuspending the stem cells by using normal saline, dyeing and counting the orchis ensiformis to obtain the stem cells, and storing the stem cells in a refrigerator at 4 ℃ for later use.

And storing part of the stem cells in a freezing storage solution consisting of 40% of stem cell culture solution, 10% of dimethyl sulfoxide and 50% of fetal calf serum in a liquid nitrogen tank at the temperature of-196 ℃ for later recovery.

the human tissue comprises waste umbilical cord, umbilical cord blood, placenta, amnion, fat, bone marrow, skin, menstrual blood, teeth, synovial fluid, synovial membrane, endometrium, brain tissue, liver tissue, pancreas, retina and the like, and is cultured in a culture device of sodium alginate gel matrix with corresponding hardness to prepare corresponding stem cells, namely umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, placental sub-totipotent stem cells, amnion stem cells, adipose-derived stem cells, bone marrow mesenchymal stem cells, bone marrow endothelial progenitor cells, epidermal stem cells, skin fibroblast stem cells, menstrual blood endometrium progenitor cells, dental pulp stem cells, synovial fluid mesenchymal stem cells, synovial membrane mesenchymal stem cells, endometrium stem cells, nerve stem cells, liver stem cells, pancreatic stem cells, retinal progenitor cells and the like.

The hypoxic culture condition is that the hypoxic concentration is 1-10%; preferably, a low oxygen concentration of 1-5% is selected. Compared with the 10 th generation stem cell cultured under the condition of low oxygen concentration, the stem cell prepared after the subculture of 30 generations has good cell morphology when observed by an inverted microscope for culturing the mesenchymal stem cell under the condition of the invention, and the phenotype of the cell detected by a flow cytometer meets the requirement; the in vitro proliferation capacity analysis shows that the compound has better proliferation activity; the stem cell colony forming experiment forms obvious stem cell clone colonies; in vitro differentiation ability analysis also indicates that it can differentiate into corresponding adult cells; preferably, in an in vivo experiment of the animal model for treating spinal cord injury by using the stem cells, the behavioral score of spinal cord injury is obviously improved, the motor function recovery of rats with spinal cord injury is promoted, the cavity repair of the rats with spinal cord injury is more facilitated, and the survival time of the stem cells transfected by the green fluorescent protein is longer in the animal in vivo experiment.

The cell culture Medium is commercially available and comprises MEM alpha and DMEM/F12 (1: 1) culture Medium of Gibco company, EBM-2 basic Medium-2 and UltraCULTURETM serum-free culture Medium of Lonza company, Neurobasal culture Medium of Gibco company, serum-free culture Medium of BIOWIT company, CHANG culture Medium of Irvine company, OriCellTM serum-free culture Medium of Cyagen Biosciences company, StemXVIVo serum-free culture Medium of R & D company, serum-free culture Medium of Thermo Fisher company and the like.

The modified sodium alginate hydrogel provided by the invention is prepared from a streptavidin-modified fibronectin-modified biotin-coupled sodium alginate hydrogel which can be combined with cytokines; a cell culture substrate material prepared by the same, a cell culture device prepared by the same, a serum-free culture additive containing soluble collagen, lactic acid, vitamin A and 2-phosphoric acid-L-ascorbic acid, and a method for culturing stem cells under the condition of low oxygen concentration. In particular, human stem cells obtained from primary culture can be prepared into biological products for pharmaceutical compositions for disease treatment by simulating the growth environment of normal stem cells in the body. In particular to a cell therapy product prepared from nervous system diseases, cardiovascular and cerebrovascular diseases, orthopedic diseases, autoimmune diseases, diabetes, nephropathy, medical plastic and the like, and is used for clinical treatment.

Specific examples of the present invention will be described below, but the technical scope of the present invention is not limited to these examples.