CN106754668B - Stem cell culture solution and injection - Google Patents
Stem cell culture solution and injection Download PDFInfo
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Abstract
A stem cell culture solution is composed of the following raw materials: 10% of compound amino acid injection, 1% of water-soluble vitamin for injection, 0.5% of alanyl glutamine, 10% of human serum albumin, 10% of hydrolyzed protein injection, 0.5% of trace element injection and 68% of Bomaili A liquid; the pH value is 6.8-7.0, and the osmotic pressure is 310 mOsm/kg. Culturing mesenchymal stem cells by using the stem cell culture solution, and collecting a mesenchymal stem cell culture supernatant; filtering the collected culture supernatant through a filter membrane, and collecting filtrate, namely the stem cell injection. The decellularized stem cell secretion factor is used for intravenous injection, so that the dispute of the stem cell in the aspect of ethics is effectively avoided; the stem cell culture solution has the advantages that the raw material components belong to clinical grades, the stem cell culture solution is safe and reliable, nutrition is provided for the survival environment of stem cells, the stem cells can be effectively promoted to secrete a large amount of cell factors, the activity of the stem cells is kept, and the sub-health state of an organism can be effectively relieved and the health of the organism can be reproduced by matching the formula of the nutrition supplement agent with the cell factors.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a mesenchymal stem cell culture solution and an injection.
Background
Mesenchymal stem cells (Mesenchymal stem cells) are a class of adult stem cells with multipotentiality, originate from early-developing mesoderm, mainly exist in connective tissues and organ interstitium, and can be obtained from various tissues such as bone marrow, peripheral blood, fat, skin and the like. Mesenchymal stem cells belong to non-terminally differentiated cells, which have characteristics of both mesenchymal cells and endothelial cells and epithelial cells; as a type of pluripotent stem cells, the pluripotent stem cells can proliferate and differentiate in the directions of bone, cartilage, muscle, tendon, liver, fat, nerve, endothelium, islet-like cells and the like under specific in vitro induction conditions.
During the culture of the mesenchymal stem cells, Stem Cell Factor (SCF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor A (PDGF-A), transforming growth factor (TGF-beta), Hepatocyte Growth Factor (HGF), Interleukin (IL), insulin-like growth factor (IGF-II), macrophage colony stimulating factor (GSFs), tumor necrosis factor (TNF-alpha), Interferon (IFN), Nerve Growth Factor (NGF), Exosome (Exosome) secreted by the cells, functional protein, mRNA and microRNA (miRNA) and other substances are secreted. The compound cell factor containing RNA molecule can promote cell proliferation, differentiation and regeneration, enhance metabolism and prevent cell aging in human body; mediating the cell signal transmission of the organism, activating the stem cells of the organism, promoting the formation of new capillary blood vessels and carrying out physiological repair; regulating sub-health of organism, improving immunity, and eliminating injured, pathological and aged cells. It has been shown that the use of stem cells to secrete factors and exosomes has the same effect as the use of stem cells. Therefore, the concept of decellularization using cell-secreted factors instead of cells in cell therapy is receiving increasing attention and support, and the advantages of stem cell-secreted factors are clear compared to stem cells.
The growth of cells in vitro depends on the culture medium, while stem cells directly secrete cytokines into the culture medium, the composition of which is directly linked to the clinical use of cytokines. At present, the in vitro culture of animal cells mainly depends on fetal calf serum, so that the cells can be well proliferated and grown in vitro. However, because the fetal calf serum has complex components, the problems of potential cytotoxicity, exogenous virus and pathogenic factor pollution exist when cells are cultured by using a serum-containing culture medium, the standardization of cell culture and the purification difficulty of a final product are increased, and the clinical anaphylactic reaction is easily caused by the residue of heterogeneous serum. Therefore, the development and application of serum-free media are imperative. The serum-free medium is a medium that does not contain animal serum or other biological extracts, but can maintain the growth and propagation of cells in vitro for a long period of time.
The world health organization refers to the state that the organism has no organic lesions but has some function changes as the third state, and the state is called as the sub-health state in China. Sub-health refers to a state of physiological hypofunction between health and disease. Clinical drugs are mostly drugs used to treat diseases, and there are no modulators directed to sub-healthy populations. For patients, some nutritional supplements are commonly used clinically to regulate the body metabolism and immune system, such as: the human serum albumin has the functions of increasing circulating blood volume and maintaining plasma colloid osmotic pressure, can be used as a nitrogen source for providing nutrition for tissues, is beneficial to cell metabolism, and can also be used as a carrier for conveying different substances; water-soluble vitamins, has effects of enhancing immunity, resisting oxidation, improving metabolism, and controlling and preventing cancer; the compound amino acid can participate in the synthesis and metabolism of protein, generate enzymes, hormones, antibodies and structural proteins, promote tissue healing and restore normal physiological functions; glutamine, a nutritional supplement, provides a nitrogen source for the body, promotes the synthesis of proteins, improves the nutritional metabolism of the body, regulates blood glucose, and can enhance the function of the immune system and resist oxidation; a trace element injection belongs to parenteral nutrition additives and maintains normal biochemical reaction in a body; the hydrolyzed protein can enter tissue cells, participate in protein anabolism, obtain positive nitrogen balance, generate enzymes, hormones, antibodies and structural proteins and promote the physiological function of tissues to be recovered to be normal; bomaili A liquid is water, electrolyte replenisher and alkalizer, and can maintain osmotic pressure and protect cells. These clinical drugs have been used clinically for many years, and their safety and effectiveness have been recognized by society.
The patent CN102920735A and the patent CN1966080A both provide a mesenchymal stem cell injection, but the components of the injection contain living cells, so that the safety and controllability of clinical use are reduced, and moreover, fetal calf serum is used when the mesenchymal stem cells are cultured, exogenous protein is introduced, so that the use risk is higher; the patent CN1618981A provides a method for preparing stem cell secretory factor, in which stem cells are genetically modified to become immortalized cells, and because of the potential risk of genetic modification, and the separation and purification of the stem cell secretory factor, the process is complicated, and the loss of the stem cell secretory factor is inevitable; patent CN102008507A provides an umbilical cord mesenchymal stem cell injection, but only human serum albumin and heparin calcium are added, the formula has a single nutrition structure, certain influence is caused on cell activity and secretion factor content, and the safety of the umbilical cord mesenchymal stem cell injection is problematic because the umbilical cord mesenchymal stem cell injection contains living stem cells; patent CN102703385A provides a mesenchymal stem cell culture solution, and it uses cell culture solution to add serum-free additive, bFGF, EGF, fetuin and antibiotic and makes stem cell culture solution, and first cell culture solution does not belong to clinical grade reagent, can not directly be applied to the human body, and secondly, adds the antibiotic in the culture solution, has allergic risk, and finally, its nutrition structure is single, can not satisfy cell nutrition, probably has certain influence to cell activity and secretion factor content.
Whether the clinical research and treatment of stem cells or the application and development of stem cell secreted factors require a large amount of mesenchymal stem cells to be amplified, so how to obtain a large amount of mesenchymal stem cells is a precondition and a key for research.
Disclosure of Invention
In order to solve the problems, the invention provides a stem cell culture solution and an injection. The injection is directly injected intravenously, and contains no living cells. The operation process is simple and reliable, the application is safe, the immunity of the organism can be effectively improved, and the health state can be recovered.
In order to achieve the aim, the invention provides a stem cell culture solution which comprises the following raw materials in percentage by volume: 10% of compound amino acid injection, 1% of water-soluble vitamin for injection, 0.5% of alanyl glutamine, 10% of human serum albumin, 10% of hydrolyzed protein injection, 0.5% of trace element injection and 68% of Bomaili A liquid.
Culturing mesenchymal stem cells by using the stem cell culture solution, and collecting a mesenchymal stem cell culture supernatant; filtering the collected culture supernatant through a 0.22 mu m filter membrane, and collecting filtrate, namely the mesenchymal stem cell injection.
The mesenchymal stem cells are adult stem cells such as umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, umbilical cord blood mesenchymal stem cells, adipose mesenchymal stem cells, bone marrow mesenchymal stem cells or dental pulp mesenchymal stem cells.
In order to achieve the above object, the present invention also provides a method for preparing a stem cell culture solution, comprising the following steps.
Step 1, taking 1% Bomaili A liquid, and dissolving water-soluble vitamins for injection to obtain a mixed liquid.
And 2, respectively and uniformly mixing the mixed solution obtained in the step 1, the compound amino acid injection, the alanyl glutamine, the human serum albumin, the hydrolyzed protein injection, the trace element injection and 67% Bo Mai solution according to a proportion to obtain the stem cell culture solution.
The pH value of the stem cell culture solution is 6.8-7.0, and the osmotic pressure is 310 mOsm/kg.
In order to achieve the above object, the present invention also provides a method for preparing a mesenchymal stem cell injection, comprising the following steps.
Step 1, collecting and processing a mesenchymal stem cell sample.
Step 2, primary extraction and culture of stem cells: separating mesenchymal tissues by a physical shearing method, and culturing mesenchymal stem cells by a tissue block adherent culture method by adopting a serum-free culture medium; and centrifuging to separate the mononuclear cells, and separating and culturing the mesenchymal stem cells in an adherent culture mode.
And 3, subculturing stem cells: when the cells are fused by more than 70%, washing twice, adding a cell digestive fluid, filtering to remove small tissue blocks, centrifuging and discarding supernatant to obtain primary mesenchymal stem cells; and (3) after the primary mesenchymal stem cells are re-suspended by a serum-free culture medium, inoculating the primary mesenchymal stem cells into a culture bottle or a culture dish, and collecting the primary mesenchymal stem cells as seed cells or continuously subculturing when the cell fusion degree reaches 80-90%.
And 4, biological and flow cytometry identification of the mesenchymal stem cells.
And 5, preparing a stem cell culture solution, replacing the mesenchymal stem cells with the culture solution, continuously culturing the mesenchymal stem cells, and collecting a mesenchymal stem cell culture supernatant.
And 6, filtering the stem cell culture supernatant collected in the step 5 through a 0.22-micrometer filter membrane, and collecting filtrate, namely the stem cell injection.
In the step 1, the sample is treated by soaking in alcohol for 1-4min, and the alcohol is filtered and sterilized by a filter membrane with the aperture of 0.22 mu m.
The serum-free culture medium is prepared by adding 10% serum substitute into DMEM/F12 basal culture medium, and L-glutamine is required to be supplemented before use.
CO is adopted in the step 2 adherent culture2An incubator; the CO is2The conditions of the incubator were: at 37 deg.C, 12% oxygen, 5% carbon dioxide, and 83% nitrogen.
The inoculation density in the step 3 is 5000-2。
The stem cell injection can be directly applied to intravenous injection of human bodies, and the dosage is 200m per patient.
The invention has the beneficial effects.
The stem cell culture solution and the injection provided by the invention simulate the hypoxia environment in the mesenchymal stem cells and are cultured by using a serum-free culture medium, when the cell fusion rate reaches 70%, the stem cell culture solution is used for continuously culturing for 2 days, the culture supernatant of the stem cells is collected, the supernatant contains stem cell secretion factors, and the stem cell injection is obtained after filtration and can be directly applied to human intravenous injection. The invention uses the decellularized stem cell secretion factor for intravenous injection, the decellularization is beneficial to relieving the uncontrollable danger of the main department after stem cell implantation, and the decellularized supernatant can not increase the risk of peripheral microvascular obstruction and can not cause tumor; the stem cell culture solution provides basic nutrition for the survival environment of stem cells, can effectively promote the stem cells to secrete a large amount of cell factors and exosomes, keeps the activity of the stem cells, and can effectively relieve the sub-health state of an organism and reappear the health of the organism by matching a nutrition supplement formula in the culture solution with the stem cell secretion factors; the stem cell culture solution provided by the invention has the advantages that the raw material components belong to clinical grades, and the stem cell culture solution can be directly used for a human body and is safe and reliable.
The cell culture environment of the invention is a low oxygen condition culture, the environment oxygen content for physiologically in vivo cell survival is 1-13%, but most of the in vitro cell culture in the prior art is completed under 21% oxygen concentration. Mesenchymal stem cells mainly grow in the environment of hypoxia in vivo, hypoxia more conforms to the physiological state of the cells, and oxygen enrichment has an injury effect on the cells. The current research shows that the low-oxygen culture can improve the proliferation capacity, the migration capacity and the adhesion capacity of the mesenchymal stem cells and reduce the cell decay rate. However, more lactic acid is produced under hypoxia, and as a metabolic byproduct, excessive lactic acid is toxic to cells and limits further cell growth, so that the present invention has been largely practiced to give 12% oxygen suitable for hypoxic conditions of mesenchymal stem cells. According to the invention, the serum substitute is adopted to replace fetal bovine serum used in the traditional mesenchymal stem cell culture, and potential risks brought by animal serum and animal-derived foreign proteins can be avoided in clinical application.
Meanwhile, the culture solution and the injection of the mesenchymal stem cells provided by the invention are suitable for large-scale industrial production and have great application prospects.
Drawings
Fig. 1 is a diagram of the growth state of mesenchymal stem cells.
Figure 2 is a flow cytometric map of mesenchymal stem cells; wherein, the expression rate of the mesenchymal stem cell CD90 is 2-1, the expression rate of the mesenchymal stem cell CD73 is 2-2, the expression rate of the mesenchymal stem cell CD105 is 2-3, the expression rate of the mesenchymal stem cell CD45 is 2-4, and the expression rate of the mesenchymal stem cell CD34 is 2-5.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1.
A preparation method of umbilical cord mesenchymal stem cell culture solution and injection comprises the following steps.
Step 1, sample collection.
Collecting fresh sterile umbilical cord tissue of a healthy fetus at term with informed consent of a puerpera, storing at 4 ℃, and treating in 24 h. Collecting maternal blood in seven days before delivery for HIV detection, hepatitis B detection, hepatitis C detection, syphilis detection, glutamic-pyruvic transaminase detection, cytomegalovirus detection and mycoplasma detection, and using fresh and sterile umbilical cord tissue of fetus after all detection is qualified.
Cleaning and disinfecting: taking out the umbilical cord from the biological safety cabinet, firstly cleaning the surface of the umbilical cord by using 10ml of 0.9% sodium chloride injection, and then immersing the umbilical cord into 75% alcohol which is subjected to filtration sterilization by 0.22 mu m for 2 min; finally, the mixture is washed again by 10ml of 0.9% sodium chloride injection to remove residual alcohol and bloodstain.
And 2, primary extraction and culture of stem cells.
(1) Cutting umbilical cord into 2-3cm segments, removing outer amnion and internal blood vessel to obtain Wharton's jelly, and cutting into 2mm pieces3The tissue small blocks are washed for 3 times by 20ml of normal saline to obtain the tissue small blocks of the Wharton jelly without bloodstains.
(2) Digesting the obtained small piece of Wharton's jelly tissue without blood stain with 10mL of collagenase type I (0.1 mg/mL) for 10min, and releasing extracellular matrix protein from the tissue to obtain a mixture consisting of the extracellular matrix protein and the small piece of tissue.
(3) Taking 2-3mL of the mixture, smearing the mixture on a culture dish with the diameter of 10cm, preferably just fully spreading the mixture on the bottom of the culture dish, and uniformly distributing small tissue blocks, and moving the positions of the small tissue blocks by using sterile forceps to uniformly distribute the small tissue blocks; and naturally airing for 2min in the biological safety cabinet to form a colloidal coating layer adhered to the bottom of the culture dish, and fixing the small tissue blocks at the bottom of the culture dish.
(4) 10ml of serum-free medium was added to the above dish and placed in CO2Culturing in an incubator at 37 deg.C under 12% oxygen, 5% carbon dioxide and 83% nitrogen under low oxygen condition, and performing primary total liquid change after 24 hr; culturing for 4-5 days, observing cell growth from the tissue small block under microscope, culturing to 8 days, performing second total liquid exchange, and collecting the tissue small block.
The serum-free medium is prepared by adding 10% serum substitute (commercially available serum substitute, PALL company, 15950-017) into DMEM/F12 basal medium, does not contain animal-derived substances, and needs to be supplemented with L-glutamine to make its concentration 15mg/L before use. The serum substitute is used for replacing fetal bovine serum used in the traditional culture of the human umbilical cord mesenchymal stem cells, and potential risks brought by animal serum and animal-derived foreign proteins can be avoided in clinical application.
(5) And (3) transferring the tissue small blocks collected in the step (4) into a new culture dish, carrying out secondary adherent culture on the tissue small blocks, culturing primary mesenchymal stem cells, and carrying out total liquid change according to the growth condition of the cells (generally culturing the cells to the 5 th-6 th day) so as to supplement nutrient substances.
Differences in tissue patch adherence and cell growth among different culture methods of tissue patches are shown in table 1. In the embodiment, when the tissue small block is adhered to the bottom of the culture dish, the culture dish is inverted when no culture medium is added, and the tissue small block does not fall from the bottom of the culture dish.
Table 1: and comparing the adhesion of small tissue blocks and the growth of cells.
As can be seen from Table 1: the bottom of the culture dish is coated with the colloidal mixture, which is beneficial to the quick adherence of small tissue blocks and can obviously promote the growth of cells from the tissue blocks and the growth of the cells at the bottom of the culture dish; in the same time, after the tissue small blocks with the same quantity are cultured by the method, the quantity of the obtained umbilical cord primary mesenchymal stem cells is doubled compared with that of the umbilical cord primary mesenchymal stem cells obtained by a common tissue block adherence method.
The mixture of the invention forms a coating layer at the bottom of the culture dish, on one hand, the wall sticking time of small tissue blocks is shortened; on the other hand, the cells are more easily dissociated from the tissue small blocks (the coating layer constructs a three-dimensional growth environment for cell growth, which not only promotes the adhesion of the cells to the bottom of the culture dish, but also is beneficial to the penetration of culture solution and the proliferation and migration of the cells at the bottom of the culture dish), so that the culture time of the primary cells is greatly shortened. Compared with the traditional enzyme digestion method, the method adopts collagenase I with mild property to digest and obtain the extracellular matrix protein in a short time, most cells are always kept in small tissue blocks, the damage of enzyme to cell membranes is avoided, and the cell activity is ensured.
The culture time and flow detection results required for the primary culture amplification of umbilical cord mesenchymal stem cells to 70% confluence under different oxygen concentrations are shown in table 2.
Table 2: and (3) performing amplification time and flow detection on umbilical cord mesenchymal stem cells under different oxygen concentrations.
As can be seen from Table 2, the umbilical cord mesenchymal stem cells cultured under the conditions of different oxygen concentrations all meet the standards of the International society for cell therapy on mesenchymal stem cells: the positive expression rate is more than or equal to 95 percent, the negative expression rate is less than or equal to 2 percent, and the cell detection results cultured under the conditions of different oxygen concentrations are consistent, which shows that the hypoxic conditions have no influence on the growth of the umbilical cord mesenchymal stem cells; however, the culture time of the umbilical cord mesenchymal stem cells is the shortest under the hypoxic condition of 12% oxygen, but the culture time becomes long when the oxygen concentration exceeds 18%, so the hypoxic condition of 12% oxygen is used in the present invention.
And 3, subculturing the stem cells.
(1) When the cells are observed by a microscope to be fused more than 70%, carrying out passage expansion: removing serum-free culture medium, washing with 0.9% sodium chloride injection for 2 times, adding 5ml Cell digestive fluid (model: ACCUTASE GMP, Innovative Cell Technologies), separating adherent cells from the bottom of the culture dish, filtering to remove tissue small pieces, centrifuging at 1000rpm for 10min, and discarding supernatant to obtain primary umbilical cord mesenchymal stem cells.
(2) The obtained primary cells are re-suspended by serum-free medium and then inoculated in a culture bottle or a culture dish, and the inoculation density is controlled at 5000-2And after culturing for a period of time, collecting the first generation of umbilical cord mesenchymal stem cells as seed cells or continuously subculturing when the cell fusion degree reaches 80% -90%.
Subcultured umbilical cord mesenchymal stem cells are cultured for 3 days under the condition of different oxygen concentrations of 6% -21%, and secreted factors in cell culture supernatant are respectively detected according to the operation of an ELISA kit (American, R & D company) specification, such as: the contents of Stem Cell Factor (SCF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), interleukin 6 (IL 6), Hepatocyte Growth Factor (HGF) and tumor necrosis factor beta 3 (TNF-beta 3) are as follows.
(1) Taking out the enzyme label plate, and respectively adding 100 mu L of cytokine standard substance in blank holes as positive control according to the sequence; respectively marking the sample numbers, adding 100 mu L of umbilical cord mesenchymal stem cell culture supernatant into blank wells, and incubating for 90min at 37 ℃.
(2) Washing the plate washer for 4 times, and standing for 10-20s each time; mu.L of biotin-labeled antibody working solution was added to each well, and incubated at 37 ℃ for 60 min.
(3) Washing the plate washer for 5 times, and standing for 10-20s each time; add 100. mu.L of enzyme conjugate working solution to each well and incubate at 37 ℃ for 30 min.
(4) Washing the plate washer for 4 times, and standing for 10-20s each time; add 100 μ L of color reagent into each well, incubate reaction for 15 min at 37 deg.C in the dark.
(5) The reaction was stopped by adding 50. mu.L of stop solution to each well.
Each sample was provided with 3 replicate wells, absorbance (a) at 450nm was measured using a full-automatic enzyme calibration instrument, the average value was taken, and cytokine content was calculated according to the plotted standard curve, with the results shown in table 3.
Table 3: and (3) measuring the content of the factors secreted by the umbilical cord mesenchymal stem cells under different oxygen concentrations (unit: pg/mL).
As can be seen from Table 3, the content of the selected 7 cytokines is determined, and it can be seen that under the hypoxia condition of 12% oxygen, the content of the cytokines secreted by the umbilical cord mesenchymal stem cells is the highest and is increased by 2-4 times, so that the yield of the cytokines can be greatly increased by culturing the cells under the hypoxia condition of 12% oxygen in the whole experiment.
And 4, biological and flow cytometry identification of the umbilical cord mesenchymal stem cells.
1. Cell growth and morphological characteristics: by adopting the digestion method in the step 1 of the embodiment, the mixture is smeared on a culture dish for adherent culture, and the growth of a plurality of adherent cells can be found by observing through an inverted microscope when the mixture is cultured to 3 to 5 days; when the culture is carried out for 10 days, the cell fusion rate reaches 70 percent, as shown in figure 1; when the tissue blocks are secondarily utilized, adherent growth of cells is observed after culturing for 24 hours, and the cell fusion rate reaches about 70% after 9 days.
2. Flow cytometry identifies mesenchymal stem cell surface markers.
Second generation cells were collected, washed 2 times with phosphate buffered saline (centrifugation at 1000rpm for 5 min), and cell concentration was adjusted to 5 × 106Per ml; adding an antibody corresponding to a cell surface marker, incubating for 30min, washing again, resuspending, and detecting on a machine (FACSCalibur, BD Co.). The antibodies used included antibodies to the following cell surface molecules: CD90, CD105 and CD73 are positive markers, and CD45 and CD34 are negative markers. The specific results are shown in table 4, and the results are shown in fig. 2.
Table 4: and (5) detecting the surface marker of the umbilical cord mesenchymal stem cells.
As can be seen from Table 4: in this example, the primary umbilical cord mesenchymal stem cells harvested from the tissue pellet still meet the mesenchymal stem cell standards of the international cell therapy society after two subcultures: the positive expression rate is more than or equal to 95 percent, and the negative expression rate is less than or equal to 2 percent; the obtained cells can be used as seed cells for clinical research and application.
And 5, preparing a stem cell culture solution, then continuously culturing stem cells by using the stem cell culture solution, and collecting a stem cell culture supernatant.
1. And preparing a stem cell culture solution.
(1) 2ml of Bomaili A solution is taken to dissolve water-soluble vitamins for injection (Huarui pharmaceutical Co., Ltd., batch number: national standard H32023002, specification 0.5 g/bottle).
(2) The mixed solution obtained in the step (1), compound amino acid injection (Beijing Pherson Eussca medicine Co., Ltd., batch number: national Standard H19993345), alanyl glutamine (Huarui medicine Co., Ltd., batch number: national Standard H20058281), human serum albumin (Hualan bioengineering Co., Ltd., batch number: national Standard S19993024), hydrolyzed protein injection (Liaoyuan Hongyuan medicine Co., Ltd., batch number: H22026405), microelement injection (Huarui medicine Co., Ltd., batch number: national Standard H32023907), Bomai A solution (Shanghai Baite medicine Co., Ltd., batch number: H20000475) are mixed according to the proportion, the pH value is 6.8-7.0, and the osmotic pressure is 310 mOsm/kg, thus obtaining the dry cell culture solution. The formulation ratio of the stem cell culture solution is shown in table 5.
(3) And (3) when the umbilical cord mesenchymal stem cells grow to 70% fusion, sucking and removing culture supernatant, washing the cells once by using normal saline, washing by using the stem cell culture solution prepared in the step (2), and then continuously culturing for 2 days by using the stem cell culture solution.
(4) The Cell state was continuously observed, and after 2 days, Cell culture supernatant was collected, and after digestion with Cell digest solution (model: ACCUTASEPGMP, manufacturer Innovative Cell Technologies), cells were collected for Cell analysis.
Table 5: the formula proportion of the stem cell culture solution.
As can be seen from Table 5: culturing umbilical cord mesenchymal stem cells by using a stem cell culture solution, measuring the cell viability rate after 2 days, and starting from a second group, keeping the stem cell viability rate to be more than 96.4%, wherein the stem cell viability rate of the second group is the highest and is 97.5%, and comprehensively considering the value, the invention selects the second group as a preferable formula of the stem cell culture solution, namely: 20ml of compound amino acid injection, 0.25g of water-soluble vitamin for injection, 1ml of alanyl glutamine, 20ml of human serum albumin, 20ml of hydrolyzed protein injection, 1ml of trace element injection and 136ml of Bomaili A solution.
(2) And (3) measuring the concentration of the factors secreted by the umbilical cord mesenchymal stem cell injection by using an ELISA method.
The concentrations of Stem Cell Factor (SCF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor A (PDGF-A), interleukin 6 (IL 6), interleukin 1 (IL 1), Hepatocyte Growth Factor (HGF), tumor necrosis factor beta 3 (TNF-beta 3) and granulocyte colony stimulating factor (GM-CSF) were selectively determined as follows.
(1) Taking out the enzyme label plate, and respectively adding 50 mu L of cytokine standard substance in blank holes as positive control according to the sequence; respectively marking sample numbers, and adding 50 mu L of the stem cell culture supernatant prepared in the step 5 into blank holes; adding 10 mu L of biotin labeling solution into the sample hole; add 50. mu.L of enzyme labeling solution to the standard wells and sample wells.
(2) Incubating at 37 ℃ for 60 min; washing the plate washer for 5 times, and standing for 20s each time; adding 50 mu L of substrate A, B solution into each hole; incubating for 15 min at 37 ℃ in a dark place; add 50. mu.L of H per well2SO4And stopping the reaction by using the stop solution.
(3) Each sample was provided with 3 replicate wells, absorbance (a) at 450nm was measured using a full-automatic enzyme calibration instrument, the average value was taken, and cytokine content was calculated according to the plotted standard curve, with the results shown in table 6.
10ml of each of the albumin physiological saline and the stem cell culture solution were used for culturing umbilical cord mesenchymal stem cells for 2 days, and then culture supernatants were collected, and the results of comparing the cytokine contents are shown in Table 6.
Table 6: cytokine content comparison (unit: pg/mL).
As can be seen from Table 6: the determination of the contents of the 10 selected cytokines shows that the contents of the cytokines in the culture supernatant of the stem cells cultured by the stem cell culture solution are improved by 2 to 4 times compared with the albumin physiological saline. Therefore, the yield of the obtained cell factors can be greatly improved by using the stem cell culture solution provided by the invention to culture the stem cells.
And 6, preparing the stem cell injection by using the collected stem cell supernatant.
(1) Filtering the collected cell supernatant through a 0.22 mu m filter membrane, and collecting filtrate, namely the stem cell injection.
(2) Bacteriological identification, and gram-negative and gram-positive bacteria detection show that the result is negative.
(3) Endotoxin detection is carried out by applying a gel method, and the endotoxin content in each 1ml is 0.2EU, which meets the requirement of national formulary on injection of 0.5 EU.
The stem cell injection is directly applied to human intravenous injection of premature ovarian failure patients, 4 times of monthly injection are carried out, the dose is 200ml per person/time, 2 months of continuous injection is carried out, premature ovarian failure symptoms of premenopausal symptoms such as hot flashes, hyperhidrosis, vaginal dryness, decreased libido and the like are obviously improved, the hot flashes and hyperhidrosis phenomena disappear, vaginal secretion is normal, and the libido is recovered to be normal.
Claims (6)
1. A preparation method of umbilical cord mesenchymal stem cell injection is characterized by comprising the following steps:
step 1, collecting and processing an umbilical cord mesenchymal stem cell sample;
step 2, primary extraction and culture of umbilical cord mesenchymal stem cells: separating mesenchymal tissues by a physical shearing method, and culturing umbilical cord mesenchymal stem cells by a tissue block adherent culture method; centrifuging and separating mononuclear cells by using lymphocyte separation liquid, and separating and culturing umbilical cord mesenchymal stem cells in an adherent culture mode; CO is adopted in the adherent culture2An incubator; the CO is2The conditions of the incubator were: low oxygen condition of 12% oxygen, 5% carbon dioxide, 83% nitrogen at 37 deg.C;
step 3, subculturing umbilical cord mesenchymal stem cells: when the cells are fused by more than 60%, washing twice, adding a cell digestive fluid, filtering to remove small tissue blocks, centrifuging and discarding supernatant to obtain primary mesenchymal stem cells; after being resuspended by serum-free medium, the primary mesenchymal stem cells are inoculated in a culture bottle or a culture dish, and when the cell fusion degree reaches 80-90 percent, the primary mesenchymal stem cells are collected and used as seed cells or continuously subcultured;
step 4, biological and flow cytometry identification of umbilical cord mesenchymal stem cells;
step 5, taking the prepared umbilical cord mesenchymal stem cell culture solution, replacing the umbilical cord mesenchymal stem cell culture solution, continuously culturing the umbilical cord mesenchymal stem cells, and collecting the culture supernatant of the umbilical cord mesenchymal stem cells;
step 6, filtering the umbilical cord mesenchymal stem cell culture supernatant collected in the step 5 through a 0.22-micrometer filter membrane, and collecting filtrate, namely the umbilical cord mesenchymal stem cell injection;
the umbilical cord mesenchymal stem cell culture solution is prepared from the following raw materials in percentage by volume: 10% of compound amino acid injection, 1% of water-soluble vitamin for injection, 0.5% of alanyl glutamine, 10% of human serum albumin, 10% of hydrolyzed protein injection, 0.5% of trace element injection and 68% of Bomaili A liquid; the pH value is 6.8-7.0, and the osmotic pressure is 310;
the serum-free medium is prepared by adding 10% serum substitute into DMEM/F12 basal medium.
2. The method for preparing umbilical cord mesenchymal stem cell injection according to claim 1, wherein the method for preparing umbilical cord mesenchymal stem cell culture solution comprises the following steps:
step 1, taking Bomaili A liquid, and dissolving the Bomaili A liquid in water-soluble vitamins for injection to obtain mixed liquid;
and 2, respectively and uniformly mixing the mixed solution obtained in the step 1, the compound amino acid injection, the alanyl glutamine, the human serum albumin, the hydrolyzed protein injection, the trace element injection and the Bo Mai LiA solution according to a proportion to obtain the umbilical cord mesenchymal stem cell culture solution.
3. The method for preparing umbilical cord mesenchymal stem cell injection as claimed in claim 1, wherein the sample is treated in step 1 by soaking in alcohol for 1-4min, and the alcohol is sterilized by filtration through a 0.22 μm pore size filter membrane.
4. The method for preparing umbilical cord mesenchymal stem cell injection as claimed in claim 1, wherein the density of the inoculation in the step 3 is 5000-6000/cm2。
5. The method for preparing umbilical cord mesenchymal stem cell injection according to claim 1, wherein the method for preparing umbilical cord mesenchymal stem cell culture solution comprises the following steps:
step 1, dissolving 1% Bomaili A liquid in water-soluble vitamin for injection to obtain a mixed solution;
and 2, respectively and uniformly mixing the mixed solution obtained in the step 1, the compound amino acid injection, the alanyl glutamine, the human serum albumin, the hydrolyzed protein injection, the trace element injection and the 67% Bo Mai liquid according to a proportion to obtain the umbilical cord mesenchymal stem cell culture solution.
6. The method for preparing umbilical cord mesenchymal stem cell injection according to claims 1 to 5, wherein the umbilical cord mesenchymal stem cell injection is applied to human intravenous injection, and the injection dosage is 200 ml/time.
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