CN103305453A - Microcarrier culture system of umbilical cord mesenchymal stem cells - Google Patents
Microcarrier culture system of umbilical cord mesenchymal stem cells Download PDFInfo
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- CN103305453A CN103305453A CN2013101936635A CN201310193663A CN103305453A CN 103305453 A CN103305453 A CN 103305453A CN 2013101936635 A CN2013101936635 A CN 2013101936635A CN 201310193663 A CN201310193663 A CN 201310193663A CN 103305453 A CN103305453 A CN 103305453A
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Abstract
The invention belongs to the field of biological medicines, and discloses a microcarrier culture system of umbilical cord mesenchymal stem cells (MSC), and preparation and applications of an MSC preserving fluid. The microcarrier culture system is characterized by preparing a heparin crosslinking sephadex microcarrier and a mating microcarrier culture medium, and a high-efficiency MSC culture system can be built. MSC cells can be efficiently and fast prepared within a short period by utilizing the microcarrier culture system of the MSC. The MSC preserving fluid is characterized by adding human albumin, heparin sodium, estrogen and IL-1beta into a ringer solution, the cell activity of the MSC cells can be kept unchanged within 24 hours by utilizing the preserving fluid, and the level of cell factors can be improved by about five times on average. The microcarrier culture system is applied to being developed into large-batch preparation of umbilical cord MSC for clinical use and laboratories, and provides a specific preserving method for MSC for treating systemic lupus erythematosus, and establishes a basis for the clinical application of the MSC.
Description
Technical field
The present invention be a kind of fast, effectively and mass-producing obtain the method for umbilical cord MSC; specifically utilize the serum-free that cell microcarrier culture technique and the special cells factor consist of as culture system, the raising of mass-producing to obtain umbilical cord mesenchymal stem cells yield, speed and quality, and develop special cell-preservation liquid according to different clinical applications.
Technical background
Mescenchymal stem cell (MSC) is to belong to a mesoblastic class multipotential stem cell, mainly be present between reticular tissue and organ in the matter, it is except having the multi-lineage potential of stem cell, also have immunoloregulation function, reduced immunogenicity and easy characteristics such as the property obtained, be subjected to extensive attention clinically at present.MSC has become the focus in the stem-cell research in recent years, has preferably application prospect in fields such as Immunological diseases treatment, hematopoietic stem cell transplantation, organizational project, genetically engineereds.But the clinical application of MSC is subject to the restriction of the technical elements such as its quantity of obtaining, quality and directed differentiation, does not also carry out at present widely clinical application.Therefore set up stable fast and effectively MSC mass-producing culture systems and have very important meaning.
It is generally acknowledged that the content of MSCs in myeloid tissue is the abundantest, but along with wearing out of age, the MSCs number significantly reduces in the autologous bone marrow, the proliferation and differentiation ability significantly fails, and isolating MSC at present from the marrow such as fat, embryo, amniotic fluid, placenta, Cord blood and umbilical cord tissue has in addition become study hotspot.Wherein umbilical cord is easy to obtain owing to it, and umbilical cord MSC cell has identical phenotype and differentiation potential with marrow MSC cell.
Method for separating of mescenchymal stem cell mainly contains 4 kinds at present, i.e. fluidic cell separating method, magnetic bead partition method, density gradient centrifugation and adherent sieve method.Wherein, adherent sieve method, the cellular form homogeneous that obtains, rate of propagation is fast, and growth conditions is stable, and easy and simple to handle, and cost is low, is expected to become practical production method.MSC has unlimited multiplication capacity in theory, but because the undifferentiated state of MSC can only be kept the limited time during vitro culture, and along with the propagation of passage number, the multiplication capacity of MSC descends, form is also become roomy flat by spindle shape, lose gradually the ability of Multidirectional Differentiation.Adherent growth is owing to being subject to the restriction of culture vessel, need repeatedly pancreatin digestion, go down to posterity and increase, process is loaded down with trivial details, easily pollutes, and the culture environment heterogeneity that causes of nutrition and products of cellular metabolism, is unfavorable for the large scale culturing of MSC.
Much studies show that at present, some cytokines can promote the propagation of cell, such as the acidic fibroblast factor, heparin, VEGF and FGF-2 etc.Although, the multiplication capacity of the MSC of the increase that these cytokines all can be in various degree, its mechanism of action is illustrated not yet fully, and very complicated on propagation and the differentiation impact of MSC, this brings difficulty for the clinical application safety evaluation.In order to increase the multiplication capacity of MSC, and do not consider after adding that its differentiation potential run counter to the clinical meaning of MSC.
1967, the Van Wezel spent ion exchange resin DEAE-SephadexA-50 that takes the lead in made microcarrier, was used for culturing cell.Microcarrier is cultivated and is compared with monolayer cell culture, amasss for attached cell provides larger culture surface, thereby the output of cell is increased greatly, and be easy to control, has reduced the danger of polluting, and makes scale operation become possibility.At present, adopt the MSC cell of micro-carriers cell culture system cultivator in stirred bioreactor, with explore a kind of in a large number, the MSC cultural method has become a kind of trend fast.Microcarrier is that diameter is at the microballon of 60～250 μ m.At present, also be not applicable to microcarrier cultural system and the special culture media thereof of MSC large scale culturing, the culture systems of other cell of cultivation and the general ordinary culture mediums that contain animal derived serum of adopting more, this has caused following problem: 1. apoptotic cell is more: this mainly is because the shearing force that micro-carrier system commonly used produces under common rotary bio-reactor stirring state is larger, and the MSC cell is caused certain damage; 2. collecting cell difficulty comparatively: because MSC is the cell of adherent growth, thus under common microcarrier culture system, need to digest collection with 0.25% pancreatin when collecting cell, and that the MSC that the microcarrier of some porous is cultivated collects is just more difficult; 3. heterology albumen: under general culture condition, in substratum, all be added with 10% foetal calf serum.2010, the developed countries such as the U.S., European Union stopped at comprehensively and use serum in the bio-pharmaceuticals.Therefore, in micro-carriers cell culture, adopt serum-free, without albumen and chemical composition defined medium, become the study hotspot of cell cultures.
We have improved traditional microcarrier culture system, adopted the microcarrier medium of novel material, and use the wave bio-reactor will lack traditional culturing process shearing force to the damage of cell, and adopted microcarrier to cultivate special serum free medium, with the loss cell that has lacked in the MSC collection process, improved greatly yield and the efficient of MSC.Present method is simple, more is conducive to for the production of practice.
Summary of the invention
The purpose of this invention is to provide a kind of microcarrier cultural system, take the quick umbilical cord of newborn fetus that effectively utilizes as the method for resource acquisition umbilical cord MSC and quick MSC evaluation.This cover microcarrier cultural system specifically comprises following content:
1, heparin sephadex microcarrier: in traditional Cytodex3 preparation of microcarriers process, increase according to denatured collagen: heparin (10: 1 ratios) mixes and the chemical coupling of sephadex pedestal.The denatured collagen layer is easily by multiple protein enzymic digestions such as trypsinase and collagenases, in order to the pancreatin collecting cell time, can when keeping cell maximum activity, function and integrity cell be separated from microcarrier.Heparin on the microcarrier surface can promote propagation and the growth of MSC cell.
2, MSC cell non-serum microcarrier substratum: its main component is the AIM-V substratum, the inside adds hFGF-2 (50ng/ml), people Midkine (60ng/ml), soybean pancreatin inhibitor (5ng/ml), penicillin (100units/ml) and Streptomycin sulphate (100units/ml).
3, MSC cell non-serum microcarrier culture condition: microcarrier density is 3g/l heparin sephadex microcarrier, and inoculum density is 0.3 * 106 cell/ml, is seeded in the bio-reactor of Cellbag-1L, with 10rpm, 6 °, Ph7.0-7.2,37 ℃, 5%CO2 is culture condition.
4, treatment is preserved liquid with MSC: this preservation liquid main component is the clinical ringer's solution of using, 1% clinically receive with heparin with the clinical of human albumin and 1000AxaIU/ml.Then the not same-action of bringing into play in various disease according to MSC adds special cytokine to safeguard or to strengthen the biological activity of MSC.Therapy for Systemic Lupus Erythematosus is preserved liquid with MSC, and (above-mentioned preservation liquid is added with 10
-7The IL-1 β of oestrogenic hormon and 50ng/ml), treating cardio-cerebral vascular disease is preserved liquid (above-mentioned preservation liquid is added with the people Midkine of 50ng/ml) with MSC and osteogenic treatment is preserved liquid (above-mentioned preservation liquid is added with Human Bone Morphogenetic Proteins-4 BMP100ng/ml) with MSC.
Description of drawings:
The comparison of Fig. 1, MSC yield: the quantity of the MSC cell that the equal volume substratum obtains under different culture systems and the comparison of time.Next shared the substratum of 1L 10 days cultivation microcarrier cultural system conditions, adopted traditional adherent culture method can obtain 1.56 * 10
8Individual cell (Fig. 1, A), and adopt traditional microcarrier cultural method, the cell culture medium that contains serum with 1L can obtain 3.17 * 10
9Individual cell (Fig. 1, B), and adopt microcarrier serum-free culture of the present invention system, the 1L substratum can obtain 5.78 * 10
9Individual cell (Fig. 1, C).
Fig. 2, different microcarrier are cultivated the comparison that the downflow system cell instrument detects apoptosis rate: adopt traditional microcarrier to cultivate and obtain cell, the apoptosis rate of cell is 15.6%, and the apoptosis rate of cell only is 5.2% under culture systems of the present invention.
Fig. 3, preserve liquid preserve cell after 24 hours the apoptosis situation detect: use cell-preservation liquid of the present invention, still keep higher activity at the state of 4 degree cell after 24 hours.A is Therapy for Systemic Lupus Erythematosus preservation liquid, and B is that treating cardio-cerebral vascular disease is that osteogenic treatment is with preserving liquid with preserving liquid and C.
Fig. 4, Therapy for Systemic Lupus Erythematosus are with after preserving liquid preservation MSC cell 24h, and MSC cellular inflammation factor mRNA level is strengthened.A is that common ringer's solution is preserved, and B preserves for using patent of the present invention to preserve liquid.
Embodiment:
The present invention has announced the mass-producing training method of a kind of umbilical cord source MSC of optimization, and its advantage is easy, quick, economical and without foreign protein, can be directly in clinical application.The below illustrates concrete utilization of the present invention with regard to specific embodiment.
Example 1 umbilical cord MSC tentatively obtains:
1. umbilical cord is selected: choose the puerpera through strict pathogen detection (microbial pathogenes such as treponema pallidum, hiv virus (HIV), cytomegalovirus (CMV), hepatitis B virus (HBV), hepatitis C virus (HCV), syphilis, mycoplasma detect), confirm to use after the safety.Clip cuts open the palace and produces nearly placenta end umbilical cord 20-30cm in puerpera's operation, places the PBS of aseptic precooling to preserve.
2. umbilical cord preliminary treatment: in Bechtop, glass culture dish is put in the umbilical cord taking-up, be cut into the approximately long segment of 5cm, after cleaning with PBS, peel off Umbilical artery and umbilical vein, and the umbilical cord tissue that will peel off blood vessel is put in the DMEM/F12 substratum and is cut into 2mm with surgical scissors
3About tissue block.
3. configure CDH digestive ferment (II Collagenase Type 250U/ml, neutral protease 100U/ml, Unidasa 10U/ml), 37 degree are dissolved in the DMEM/F12 substratum, and the filter degerming of filtering 0.22 μ m is for subsequent use.Digestive ferment is preferably now with the current, spends the shelf time above 1 week 4.
4. the tissue that shreds and CDH mixed enzyme solution by 1: 1 volume ratio mix with the 50ml centrifuge tube in, place 37 ℃ of shaking tables, 200rpm vibration is about digestion 3h (treat tissue block digests substantially get final product, the residual MSC that do not affect of the tissue block that minority is less obtains efficient).
5. with 4 ℃ in postdigestive tissue juice, the centrifugal 5min of 300g abandons supernatant, abandons supernatant, will precipitate with PBS to be resuspended in the 50ml fresh culture, and 4 ℃, the centrifugal 5min of 300g abandons clearly, and PBS washes twice, notes cell and tissue precipitation not being discarded.Last abandons supernatant all over after centrifugal, precipitation is resuspended in the DMEM/F12 substratum that contains 12% foetal calf serum, spreads the T-25 culturing bottle.
6. when treating that cell grows to 80% abundance (approximately 8 days), with cell dissociation buffer (0.25% pancreatin+0.02%EDTA), collect cell.
The microcarrier serum-free culture of example 2 umbilical cord MSC:
1, microcarrier is processed: heparin sephadex microcarrier washs 3 times in the PBS that does not contain Ca2+ and Mg2+ ion after, and at 121 ℃ of autoclaving 15min, and at 4 ℃ of lower Preservation in sterile conditions.Microcarrier is first with the substratum washing before using, and transfers in front 24 hours in inoculation and to carry out balance among the bio-reactor Cellbag.In order to prevent that microcarrier is adsorbed onto glass surface, the transfer bottle of all uses and rolling bottle all carry out siliconizing.
2, microcarrier serum-free culture: microcarrier density is 3g/l, and inoculum density is 0.3 * 10
6Cell/ml is seeded in the bio-reactor of Cellbag-0.5L, with 10rpm, and 6 °, Ph7.0-7.2,37 ℃, 5%CO2 is culture condition.24 hours, behind the cell attachment, speed of rotation changed 30rpm into, and 7 °, other condition is constant, and amount was changed liquid in per 2 days half.
3, cell harvesting: sampling every day, Viola crystallina is observed, and about 10 days, after cell covers with, stops the rotation.After covering with the microcarrier sedimentation of cell, collect with PBS, after the washed twice, with collecting cell after 0.25% the trysinization, and with the PBS cleaning for future use.
Example 3 treatments are preserved liquid with MSC and are used:
1, three kinds of preparations of preserving liquid: in strict accordance with aseptic technique, get the clinical ringer's solution 250ml that uses, add and clinically receive with heparin with human albumin 2.5ml and the clinical of 250,000 AxaIU/ml, be equally divided into three parts behind the mixing, every part of 80ml.In every part, add respectively respectively: 10
-7The people Midkine (treating cardio-cerebral vascular disease is used) of the IL-1 β of M oestrogenic hormon and 50ng/ml (Therapy for Systemic Lupus Erythematosus is used), 50ng/ml and the Human Bone Morphogenetic Proteins-4 BMP (osteogenic treatment is used) of 100ng/ml.
2, preserve adding MSC cell in the liquid at every part respectively, cell concn is 5 * 10
6Individual cell/ml.Preserve after 24 hours for 4 ℃, flow cytometer detects the apoptosis situation.
3, preserve the expression that liquid is preserved Cox-2, IL-8 behind the 24h, IL-6 and the CXCR4 factor with Q-PCR method detection system lupus erythematosus zhiliao with MSC.
Claims (4)
1. a heparin sephadex microcarrier is characterized in that in traditional Cytodex3 preparation of microcarriers process, increases according to denatured collagen/heparin (10: 1 ratios) to mix and the chemical coupling of sephadex pedestal.
2. a micro-carriers cell culture system is characterized in that using microcarrier claimed in claim 1, and density is 3g/l heparin sephadex microcarrier, and inoculum density is 0.3 * 10
6Cell/ml is seeded in the bio-reactor of Cellbag-1L, with 10rpm, and 6 °, Ph7.0-7.2,37 ℃, 5%CO2 is culture condition.
3. MSC cell non-serum microcarrier substratum: its main component is the AIM-V substratum, the inside adds hFGF-2 (50ng/ml), people Midkine (60ng/ml), soybean pancreatin inhibitor (5ng/ml), penicillin (100units/ml) and Streptomycin sulphate (100units/ml).
4. a Therapy for Systemic Lupus Erythematosus is preserved liquid with MSC, and it mainly becomes and is the 99% clinical ringer's solution of using, 1% clinical human albumin of using, and 1000AxaIU/ml is clinical to receive 10 with heparin
-7The IL-1 β of oestrogenic hormon and 50ng/ml.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104523753A (en) * | 2015-01-22 | 2015-04-22 | 中国科学院广州生物医药与健康研究院 | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer |
| CN106479969A (en) * | 2016-10-13 | 2017-03-08 | 博雅干细胞科技有限公司 | Method using HLA G positive mescenchymal stem cell systemic lupus erythematosus |
| CN111534489A (en) * | 2020-04-29 | 2020-08-14 | 清华大学 | T lymphocyte amplification method based on 3D printing |
| CN113502261A (en) * | 2021-07-16 | 2021-10-15 | 上海市东方医院（同济大学附属东方医院） | Large-scale 3D (three-dimensional) hypoxia mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
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Cited By (6)
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| CN104523753A (en) * | 2015-01-22 | 2015-04-22 | 中国科学院广州生物医药与健康研究院 | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer |
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| CN111534489A (en) * | 2020-04-29 | 2020-08-14 | 清华大学 | T lymphocyte amplification method based on 3D printing |
| CN113502261A (en) * | 2021-07-16 | 2021-10-15 | 上海市东方医院（同济大学附属东方医院） | Large-scale 3D (three-dimensional) hypoxia mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
| CN113502261B (en) * | 2021-07-16 | 2023-08-29 | 上海东方星际干细胞科技有限公司 | Large-scale 3D low-oxygen mesenchymal stem cell culture system capable of efficiently differentiating into lipid |
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Application publication date: 20130918 |