CN115851590A - Method for purifying stem cell derivatives in large scale and application - Google Patents
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Abstract
The invention discloses a method for purifying stem cell derivatives in a large scale, and belongs to the technical field of biological cell purification. The technical scheme realizes the large-scale preparation of the derivative with high quality requirements of no serum, no animal source component, no culture medium component, high purity, high growth factor concentration and high exosome content. The purification method realizes the aim of large-scale and batch production, performs quality control at key nodes, and ensures the uniformity, safety and stability of product quality. The freeze-dried powder product obtained by the derivative can effectively prolong the storage life. The product has high concentration of effective substances, and is helpful for promoting recovery of body function.
Description
Technical Field
The invention belongs to the technical field of biological cell purification, and relates to a method for purifying stem cell derivatives in a large scale and application thereof.
Background
Stem cell derivatives are substances produced by paracrine action of stem cells, also called secretory groups, which perform various biological functions, such as anti-inflammatory, anti-apoptotic, regulating immunity, promoting tissue regeneration, etc., and the executives of these functions are mainly soluble secretory factors and exosomes.
The soluble factors in stem cell derivatives mainly include VEGF, HGF, FGF, IGF-1, IL-1, PGE2, IDO, TGF beta 2, etc. These factors are involved in cell life activities such as angiogenesis, immunoregulation, cell proliferation, apoptosis, etc.
Exosomes (exosomes) are tiny membrane vesicles secreted by most cells, with lipid bilayer membrane structures, approximately 40-100 nm in diameter.
Most of the derivatives in the current market have low purity and small preparation scale, and have a series of problems of high storage condition requirement, short shelf life and the like.
Disclosure of Invention
The first purpose of the invention is to provide a method for purifying stem cell derivatives on a large scale, the technical scheme realizes the large-scale preparation of high-quality derivatives, and the derivative products have the characteristics of no animal source components, no culture medium components, high purity, high growth factor concentration and high exosome content.
The second purpose of the invention is to provide the purification method which realizes the aim of large-scale and batch production, and performs quality control at key nodes to ensure the uniformity of product quality.
The third purpose of the invention is to provide a freeze-dried powder product of the derivative, which greatly reduces the storage requirement and can effectively prolong the storage life.
The invention is realized by the following technical scheme:
a method for purifying stem cell derivatives in a large scale comprises the following steps:
s1, culturing and amplifying the stem cells obtained by separation in a serum-free culture medium, freezing and storing, establishing a cell bank and carrying out characteristic detection;
s2, selecting and recovering cells from the cell bank, culturing to 80% confluence by using a serum-free culture medium, and collecting stem cell derivatives by using a derivative collection culture medium;
s3, carrying out step-by-step centrifugation and filtration on the collected stem cell derivatives, and removing cell debris and large particles;
s4, removing culture medium components after tangential flow concentration treatment and cleaning treatment to obtain derivative concentrated solution, and subpackaging for storage or respectively performing freeze-drying treatment on the concentrated solution to obtain derivative freeze-dried powder for storage;
the derivatives include exosomes and soluble cytokines.
A large amount of cells meeting the requirements are cultured and amplified by a serum-free culture medium and then are frozen and stored. After the quality detection is qualified by using the characteristic detection indexes, the cells can be thawed and revived for subsequent product preparation.
The characteristic tests are carried out to ensure that the cultured cells are qualified in quality, are the types of the required stem cells, and have no contamination of bacteria, viruses and the like.
The cell recovery process is conventional operation, and comprises the steps of thawing in a water bath at 37 ℃, cleaning to remove the preservation solution, and culturing in a culture medium.
The step-by-step centrifugation of the collected stem cell derivatives is to remove cell debris with large volume, facilitate filtration and avoid blocking the filter membrane. A filtration step is then used to remove small cell debris. Because cell debris inevitably occurs during the cell culture process.
The tangential flow is a concentration technique that removes excess small molecular weight species from the solution, thereby increasing the concentration of the solution.
Preferably, the stem cells include umbilical cord tissue, adipose tissue or placenta tissue-derived mesenchymal stem cells, neural stem cells
Any one of cells, epidermal stem cells, hair follicle stem cells and epithelial stem cells.
The stem cells comprise all tissues from which isolated tissues are obtained, and all stem cells are cultured in vitro.
Preferably, in step S1, the culture and amplification in the serum-free medium is performed by using a monolayer/multilayer cell factory or bioreactor, and the cells entering the cell bank are frozen in a serum-free freezing medium;
the culture conditions are as follows: low oxygen condition with oxygen partial pressure of 3-5%.
The above cultivation using a monolayer/multilayer cell factory or bioreactor is intended to expand the production scale and ensure cell quality uniformity.
Preferably, the characteristic test comprises stem cell flow test, virus test, sterility test, endotoxin test and mycoplasma test;
the stem cell flow detection is to detect CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR, nestin and sox 2;
the virus detection is to detect HBV, HCV, HCMV, EBV, HIV, B19, HPV, HHV6/7/8 and HTLV.
Preferably, in step S2, the derivative collection medium is a basal medium containing carbachol;
under the hypoxia condition with the oxygen partial pressure of 3-5%, the culture time is 20-50h;
the basic culture medium is any one of BME, DMEM and alpha-MEM;
the concentration of the carbachol is 0.01 mu mol/L-6 mmol/L. The concentration of carbachol is preferably 2.6. Mu. Mol/L.
Preferably, in the step S3, the step-by-step centrifugation is to collect the supernatant and remove the cell debris by 1500g and 10000g centrifugation in sequence at 4 ℃, and then to filter the supernatant by using 1.2 μm, 0.45 μm and 0.22 μm filtration membranes in sequence.
Preferably, in the step S4, the tangential flow concentration treatment is to utilize a tangential flow membrane to trap substances with molecular weight larger than 5 kD.
Preferably, in step S4, the derivative concentrated solution obtained in the freeze-drying process is selected, and is added with mannitol, trehalose, sodium hyaluronate and albumin for mixing and then is subjected to freeze-drying process;
50-70 wt% of the derivative concentrated solution, 5-10 wt% of mannitol, 5-10 wt% of trehalose, 5-10 wt% of albumin and the balance of sodium hyaluronate.
The raw materials are added before the freeze-drying treatment step and are used as freeze-drying protective agents and plastic agents, so that protein inactivation caused by direct freeze-drying is avoided.
Preferably, the conditions of the lyophilization process are: pre-freezing at-80 deg.C for 10-18h, placing in a freeze dryer with vacuum degree of less than 80mT, freeze drying at-50 deg.C for 20-32h, and taking out to obtain derivative lyophilized powder.
It is conventionally recommended to fill vials first and then freeze-dry them for ease of packaging and to reduce product loss.
The application of the obtained derivative concentrated solution in preparing cosmetics, or preparing raw materials of culture medium, or preparing intermediate additives of medical instruments, or preparing additives of medicines is disclosed.
Compared with the prior art, the invention at least has the following technical effects:
the invention provides a method for purifying stem cell derivatives in a large scale, and the technical scheme realizes the large-scale preparation with high quality requirements of no serum, no animal source component, no culture medium component, high purity, high growth factor concentration and high derivative content.
The purification method realizes the aim of large-scale and batch production, performs quality control at key nodes, and ensures the uniformity, safety and stability of product quality.
The freeze-dried powder product in the derivative obtained by the purification method can effectively prolong the storage life. And the concentration of the effective substances in the product is high, which is helpful for promoting the recovery of the body function.
Drawings
FIG. 1 is a schematic representation of the morphology of mesenchymal stem cells of a cell factory;
FIG. 2 is a diagram of the result of flow detection of the mesenchymal stem cell surface marker CD 105;
FIG. 3 is a diagram of the result of flow detection of the mesenchymal stem cell surface marker CD 90;
FIG. 4 is a diagram of the result of flow detection of the mesenchymal stem cell surface marker CD 73;
FIG. 5 is a diagram of the flow detection result of all negative markers of mesenchymal stem cells;
FIG. 6 is a schematic diagram of western blot detection of the expression of derivative lyophilized powder CD9, CD63 and CD 81;
FIG. 7 is a diagram showing the results of flow detection of the surface marker CD105 of adipose-derived stem cells;
FIG. 8 is a diagram showing the results of flow detection of the surface marker CD90 of adipose-derived stem cells;
FIG. 9 is a diagram showing the results of flow detection of the surface marker CD73 of adipose-derived stem cells;
FIG. 10 is a diagram showing the results of flow detection of negative markers for adipose-derived stem cells;
FIG. 11 is a diagram showing the result of flow assay of neural stem cell marker nestin;
fig. 12 is a diagram showing the flow detection result of the neural stem cell marker sox 2.
Detailed Description
Embodiments of the present invention will be described in detail with reference to the following examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the present invention, and that the specific conditions not specified in the examples are conducted under conventional conditions or conditions suggested by the manufacturer, and that reagents or equipment not specified by the manufacturer are all conventional products which can be obtained by commercial purchase.
Example 1: purification of umbilical cord mesenchymal stem cell derivatives
1. Stem cell isolation and banking
(1) Umbilical cord mesenchymal stem cells were isolated, primary cultured in 10T 75 flasks in total, and cell culture was performed using serum-free medium.
(2) Cells were passaged to P1, and 12 monolayers of the cell factory were co-seeded.
(3) And (3) when the P1 generation mesenchymal stem cells grow to 80% confluence, establishing a cell bank by adopting serum-free freezing medium for freezing, and completing quality verification of the cell bank.
(4) Cell bank cryopreserved cells 5X 10 6 And one cell, and 120 cells are frozen and stored together.
2. Cell recovery, expansion and derivative collection
(1) Resuscitating 1 cryopreserved cell, inoculating into 1 monolayer cell factory, counting as P2, culturing at 5% 2 ，5%CO 2 37 ℃ and saturated humidity.
(2) P2 cells were grown to 80% confluence, passaged to P3, and seeded with 1 ten-layered cell factory.
(3) When P3 cells were grown to 80% confluence, passage was to P4, 12 ten-layered cell factories were seeded, and 3T 75 flasks were simultaneously seeded as P4 quality check cells.
(4) When P4 cells grew to 80% confluence, cells in T75 flasks were digested and collected for flow detection to determine that the cells were mesenchymal stem cell phenotype, and the cell culture medium was changed to DMEM containing carbachol in ten-layered cell factories to collect derivatives, using 500mL of medium per cell factory.
FIG. 1 shows a morphological diagram of cells cultured in a cell factory.
As shown in FIG. 2, the CD105-APC-A subset results are shown schematically for flow assay, and the CD105 positivity is shown to be 98.5%. The above CD105-APC-A subset refers to: CD 105-allophycocyanin-aera assay-subgroup. CD105 is the name of a surface molecule, and indicates an antibody using this surface molecule of CD 105. APC (allophycocyanin), meaning that CD105 flow antibody-conjugated fluorescein is APC; a represents an analytical method, namely the abbreviation of aera; subset, representing the CD105-APC-A Subset. Namely: CD105-APC-A subset expression means: and (3) the CD105 is coupled with the flow antibody of the APC fluorescein, the target cell is marked, the flow cytometry detection is carried out, and the proportion of the CD105 positive cell subset is obtained by adopting an aera analysis method.
As shown in FIG. 3, the results of CD90 PE-A subset are shown schematically in the flow chart, which shows that the positive rate of CD90 is 98.5%.
As shown in FIG. 4, the results of CD73 FITC-A subset for flow assay are shown schematically, and the positive rate of CD73 is 99.0%.
The CD90 PE-A subset refers to: CD 90-phycoerythrin-aera assay-subgroup. CD90 is the name of a surface molecule, and indicates an antibody using CD90 as the surface molecule. PE (phycoerythrin), indicating that the CD90 flow antibody conjugated fluorescein is PE; a represents an analytical method, which is an abbreviation of aera; subset, represents this Subset of CD90 PE-A. Namely: CD90 PE-Asubset expression means: and (3) labeling the target cells by using a flow antibody of the CD90 coupled PE fluorescein, detecting by using a flow cytometry, and obtaining the proportion of the CD90 positive cell subset by using an aera analysis method.
The CD73 FITC-A subset refers to: CD 73-fluorescein isothiocyanate-aera assay-subgroup. CD73 is the name of a surface molecule, and indicates an antibody using the CD73 surface molecule. FITC (fluorescein isothiocyanate), fluorescein indicating CD73 flow antibody coupling is FITC; a represents an analytical method, which is an abbreviation of aera; subset, representing the CD73 FITC-A Subset. Namely: CD73 FITC-A subset expression means: and (3) the CD73 is coupled with a flow antibody of FITC fluorescein, target cells are labeled, flow cytometry detection is carried out, and the proportion of the CD73 positive cell subset is obtained by adopting an aera analysis method.
As shown in FIG. 5, which is a schematic representation of the results of all negative markers in flow assay, the total expression rate of all negative markers (CD 11b, CD19, CD34, CD45 and HLA-DR) is 1.31%.
(5) After 48 hours of culture, 6000mL of conditioned medium containing the stem cell derivatives was collected, and subjected to gradient centrifugation using 1500g and 10000g in order, followed by three-stage filtration using 1.2 μm, 0.45 μm and 0.22 μm filters, to obtain 5900mL of a stem cell derivative solution.
(6) The stem cell derivative solution was ultrafiltered using a 5kD tangential flow membrane and washed twice with physiological saline to obtain 59mL of a stem cell derivative concentrate.
(7) Taking 1mL of the stem cell derivative concentrated solution to complete quality detection (secretion factors, derivatives and culture medium residues), adding the rest 58mL of the derivative concentrated solution into a freeze-drying agent (60% of the cell derivative concentrated solution, 10% of mannitol, 10% of trehalose, 5% of albumin and the balance of sodium hyaluronate), and subpackaging into 290 penicillin bottles.
(8) Freeze-drying is completed according to the program (after pre-freezing for 12h at-80 ℃, placing in a freeze dryer with the vacuum degree of less than 80mT, and taking out after freeze-drying for 24h at-50 ℃) to obtain 290 stem cell derivative freeze-dried powders.
(9) As shown in fig. 6, the western blot detection of CD9 and expression derivatives of CD63 and CD81 is a schematic diagram of the detection of growth factors and derivatives of stem cell derivatives, and the results of the growth factor concentration ELISA detection of VEGF:225 ng/branch; HGF:155 ng/tube, and the recovery rate is 83.6 percent.
According to the process, 290 × 120=34800 lyophilized stem cell derivative powder can be obtained from one umbilical cord.
Example 2: adipose-derived stem cell purification
(1) The adipose-derived stem cells are extracted from adipose tissues, and the processes of culturing, establishing a bank, detecting, collecting derivatives and purifying the adipose-derived stem cells are the same as those of the umbilical cord mesenchymal stem cells, wherein about 150 cells in the bank can be frozen and stored in every 100g of adipose tissues.
(2) Growth factor concentration in derivatives ELISA test results VEGF:347 ng/branch; HGF:138 ng/tube, and the recovery rate is 78.9%.
According to the process, about 43500 adipose stem cell derivative freeze-dried powder can be prepared per 100g adipose tissues.
Fig. 7 is a graph showing the flow detection result of the adipose-derived stem cell surface marker CD105, wherein the positive rate of CD105 is 99.9%.
Fig. 8 is a graph showing the result of flow detection of the adipose-derived stem cell surface marker CD90, in which the CD90 positive rate is 100%.
Fig. 9 is a graph showing the flow detection result of the surface marker CD73 of the adipose-derived stem cells, wherein the positive rate of CD73 is 100%.
FIG. 10 is a graph showing the results of flow detection of negative markers for adipose-derived stem cells, in which the expression rate of all negative markers is 0.26%.
Example 3: neural stem cell derivative purification
(1) Extracting neural stem cells from brain tissues, adopting a neural stem cell serum-free culture medium to amplify and store the neural stem cells, wherein 50 cells in the stored cells can be frozen in each brain tissue.
(2) And performing subculture amplification and derivative collection of the neural stem cells by referring to an adipose mesenchymal stem cell culture method.
(3) Growth factor concentration in derivatives ELISA test results VEGF:140 ng/branch; HGF:233 ng/tube, and the recovery rate is 76.7%.
According to the process, about 14500 lyophilized powder of the ramification of the bronchial nerve stem cell can be prepared per 100g of adipose tissue.
FIG. 11 is a graph showing the results of flow-type assay of Nestin, which is a neural stem cell marker, and the Nestin positive rate is 96.5%.
Fig. 12 is a flow detection result chart of a neural stem cell marker Sox2, and the Sox2 positive rate is 97.0%.
Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for purifying stem cell derivatives on a large scale is characterized by comprising the following steps:
s1, culturing and amplifying the stem cells obtained by separation in a serum-free culture medium, freezing and storing, establishing a cell bank and carrying out characteristic detection;
s2, after selecting and recovering cells from the cell bank, culturing the cells to 80% confluence by using a serum-free culture medium, and collecting stem cell derivatives by using a derivative collection culture medium;
s3, removing cell debris and large particles after carrying out step-by-step centrifugation and filtration on the collected stem cell derivatives;
s4, removing culture medium components after tangential flow concentration treatment and cleaning and repairing to obtain a derivative concentrated solution, and performing subpackage storage or performing freeze-drying treatment on the concentrated solution respectively after subpackage to obtain derivative freeze-dried powder for storage;
the derivatives include exosomes and soluble cytokines.
2. The method for large-scale purification of stem cell derivatives according to claim 1, wherein the stem cells comprise any one of umbilical cord tissue, adipose tissue or placenta tissue-derived mesenchymal stem cells, neural stem cells, epidermal stem cells, hair follicle stem cells and epithelial stem cells.
3. The method for large-scale purification of stem cell derivatives according to claim 1, wherein in S1, culture and amplification are performed in a serum-free culture medium by using a single-layer/multi-layer cell factory or a bioreactor, and cells entering a cell bank are frozen in a serum-free freezing medium;
the culture conditions are as follows: low oxygen condition with oxygen partial pressure of 3-5%.
4. The method for large-scale purification of the stem cell derivative according to claim 1, wherein the characteristic detection comprises stem cell flow detection, virus detection, sterility detection, endotoxin detection and mycoplasma detection;
the stem cell flow detection is to detect CD73, CD90, CD105, CD11b, CD19, CD34, CD45, HLA-DR, nestin and sox 2;
the virus detection is to detect HBV, HCV, HCMV, EBV, HIV, B19, HPV, HHV6/7/8 and HTLV.
5. The method for large-scale purification of stem cell derivatives according to claim 1, wherein in S2, the derivative collection medium is a basal medium containing carbachol;
under the hypoxia condition with the oxygen partial pressure of 3-5%, the culture time is 20-50h;
the basic culture medium is any one of BME, DMEM and alpha-MEM;
the concentration of the carbachol is 0.01 mu mol/L-6 mmol/L.
6. The method for large-scale purification of stem cell derivatives as claimed in claim 1, wherein in the step-wise centrifugation in S3, 1500g and 10000g of centrifugation are sequentially adopted to collect supernatant and remove cell debris at 4 ℃, and then 1.2 μm, 0.45 μm and 0.22 μm of filtration membrane are sequentially adopted to filter.
7. The method for large-scale purification of stem cell derivatives as claimed in claim 1, wherein in the S4, the tangential flow concentration treatment is to use a tangential flow membrane to trap substances with molecular weight of more than 5 kD.
8. The method for large-scale purification of the stem cell derivatives according to claim 1, wherein in S4, the derivative concentrated solution obtained in the freeze-drying process is added with mannitol, trehalose, sodium hyaluronate and albumin and then subjected to freeze-drying;
50-70 wt% of the derivative concentrated solution, 5-10 wt% of mannitol, 5-10 wt% of trehalose, 5-10 wt% of albumin and the balance of sodium hyaluronate.
9. The method for large-scale purification of stem cell derivatives as claimed in claim 7, wherein the conditions of the lyophilization process are as follows: pre-freezing at-80 deg.C for 10-18h, placing in a freeze dryer with vacuum degree of less than 80mT, freeze drying at-50 deg.C for 20-32h, and taking out to obtain derivative lyophilized powder.
10. Use of a concentrated or lyophilized derivative powder obtained by the method according to any one of claims 1 to 9 for the preparation of an intermediate additive for cosmetics, culture medium, medical device, or drug.
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