CN107164325B

CN107164325B - The preparation method and kit of the oligodendroglia in the source MSCs

Info

Publication number: CN107164325B
Application number: CN201710508337.7A
Authority: CN
Inventors: 张洪钿; 苑春慧
Original assignee: Beijing Regenerated Biological Science And Technology Research Institute Co Ltd
Current assignee: Beijing Regenerated Biological Science And Technology Research Institute Co Ltd
Priority date: 2017-06-28
Filing date: 2017-06-28
Publication date: 2019-09-17
Anticipated expiration: 2037-06-28
Also published as: CN107164325A

Abstract

The invention discloses a kind of oligodendroglia reagent preparation boxes in source MSCs, including OPCs serum-free basal medium, OPCs culture additive 1, OPCs culture additive 2 and OPCs culture surface coating buffer, wherein, the OPCs serum-free basal medium is to contain B27, N2, L-Glutamine, 1,25- dihydroxyvitamin D3, triiodothyronine, the DMEM/F12 culture medium of people's epiphysin；The OPCs culture additive 1 is the DMEM/F12 culture medium containing recombination human basic fibroblast growth factor, recombinant human epidermal growth factor；The OPCs culture additive 2 is the DMEM/F12 culture medium containing recombination human platelet derived growth factor AA, recombinant human nerve trophic factors 3；The OPCs culture surface coating buffer is the DMEM/F12 culture medium containing recombined human Laminin lens, recombined human vitronectin.

Description

The preparation method and kit of the oligodendroglia in the source MSCs

Technical field

The present invention relates to cell preparation techniques field, it particularly relates to a kind of oligodendroglia in the source MSCs Preparation method and kit.

Background technique

A variety of neurodegenerative diseases such as multiple sclerosis, leukaemia, malnutrition and central lesion Deng that all myelin can be caused to lose, lack effective treatment means at present.Oligodendrocyte precursor cells (oligodendrocyte Precursor cells, OPCs) differentiation generation oligodendroglia forms sheath structure, exception in central nervous system It will lead to demyelinating disease of central nervous system change, then lead to neure damage and mental disorder, be important therapy target. Oligodendrocyte precursor cells replacement therapy or Regeneration and Repair treatment are the aixs cylinders for maintaining and repairing damage, rebuild aixs cylinder electrical conduction function The latest developments and most promising treatment means of energy.

The preparation of oligodendroglia in the prior art using embryonic stem cell (embryonic stem cells, ESCs), the acquisition of (induced pluripotent stem cells, iPSCs) vitro differentiation is induced multi-potent stem cell.ESCs comes The oligodendroglia cell preparation in source is related to serious ethics problem and in vivo the bad clinical risk of transplanting tumorigenesis, The oligodendroglia preparation in the source iPSCs is related to foreign gene transfection and more serious internal the serious of transplanting oncogenicity faces Bed risk.And ESCs or iPSCs Differentiation Induction in vitro prepares OPCs complex process, it is right using animal source components such as serum The OPCs quality of the pharmaceutical preparations and clinical safety generate unpredictable influence, while limiting OPCs preparation yield, affect clinic Research and industrialization Transformation Potential.

For the problems in the relevant technologies, currently no effective solution has been proposed.

Summary of the invention

For above-mentioned technical problem in the related technology, the present invention proposes a kind of system of the oligodendroglia in source MSCs Preparation Method and kit had both avoided the oligodendroglia cell preparation using the source ESCs and the serious ethics that generates Problem and in vivo the bad clinical risk of transplanting tumorigenesis also avoid generating using the oligodendroglia preparation in the source iPSCs Foreign gene transfection and more serious internal transplanting oncogenicity bad clinical risk, easy to operate, OPCs high income, without dynamic Material resource ingredient.

To realize the above-mentioned technical purpose, the technical scheme of the present invention is realized as follows:

A kind of oligodendroglia reagent preparation box in the source MSCs, including OPCs serum-free basal medium, OPCs training Support additive 1, OPCs culture additive 2 and OPCs culture surface coating buffer, wherein

The OPCs serum-free basal medium is to contain B27, N2, L-Glutamine, 1,25- dihydroxyvitamin D3, three Iodine desiodothyroxine, the DMEM/F12 culture medium of people's epiphysin；

The OPCs culture additive 1 is to grow containing recombination human basic fibroblast growth factor, recombinant human epidermal The DMEM/F12 culture medium of the factor；

OPCs culture additive 2 be containing recombination human platelet derived growth factor AA, recombinant human nerve nutrition because The DMEM/F12 culture medium of son 3；

The OPCs culture surface coating buffer is the DMEM/F12 containing recombined human Laminin lens, recombined human vitronectin Culture medium.

Further,

The OPCs serum-free basal medium contains 1 × B27,1 × N2,2mM L-Glutamine, 1uM 1,25- dihydroxy Vitamine D3,40ng/mL triiodothyronine, 0.5ug/mL people's epiphysin；

OPCs culture additive 1 be containing 1 ug/mL recombination human basic fibroblast growth factor, 1 ug/ The DMEM/F12 of mL recombinant human epidermal growth factor；

The OPCs culture additive 2 is to recombinate containing 1 ug recombination human platelet derived growth factor AA, 100ng/mL The DMEM/F12 of people's neurotrophin 3；

The OPCs culture surface coating buffer is to contain 100ng/mL recombined human Laminin lens, 100ng/mL recombined human glass The even DMEM/F12 of albumen.

According to another aspect of the present invention, a kind of oligodendroglia preparation method in source MSCs is provided, including such as Lower step:

S1. it is coated with culture surface 1 hour with OPCs culture surface coating buffer room temperature, inhales and abandon coating buffer, rinse postposition through PBS It saves and is no more than 1 week in 4 DEG C；

The MSCs in S2.P2 generation presses 6000/cm²Inoculation is cultivated with the MSCs containing 10% non-animal derived ingredient serum replacement Base culture converges to 60-70%；

S3. the OPCs serum-free basal medium of the culture additive 1 containing OPCs is changed, culture to 80-90% converges, with pancreas egg White enzyme solutions digestion harvest cell, is denoted as neural epithelium pre-induced cell；

S4. nerve is resuspended with the OPCs serum-free basal medium for cultivating additive 1 and OPCs culture additive 2 containing OPCs Epithelium pre-induced cell, adjustment cell density to 1.5 × 10⁴/cm², it is seeded to by the coated culture of OPCs culture surface coating buffer Surface, passage when culture converges to 60-80%, is denoted as P0 for OPCs.

Further,

The OPCs culture surface coating buffer is the OPCs culture surface coating buffer that 10 times are diluted through DMEM/F12；

OPCs culture 1 content of additive is 2% in the OPCs serum-free basal medium of the culture additive 1 containing OPCs；

It is described to cultivate OPCs training in the OPCs serum-free basal medium of additive 1 and OPCs culture additive 2 containing OPCs It is 2% that the content for supporting additive 1, which is the content that 2%, OPCs cultivates additive 2,.

It further, further include following steps after the step S4: by P0 for OPCs routinely attached cell propagating method Passage harvests P2 for cell.

Further, the inoculum density in the conventional attached cell propagating method succeeding generations is 1.5 × 10⁴/cm², training The system of supporting is that the OPCs serum-free basal medium of additive 1 and 1% OPCs culture additive 2 is cultivated containing 1%OPCs；Cultivate table Face is through the coated culture surface of OPCs culture surface coating buffer.

Further, the MSCs derives from people's umbilical cord, amnion, amniotic fluid, placenta, Cord blood, palace film, dental pulp, marrow, skin Skin, tendon, skeletal muscle.

Beneficial effects of the present invention: the present invention provides the methods for preparing oligodendroglia as source using MSCs, and mention A kind of kit that oligodendroglia is prepared using MSCs as source is supplied.MSCs is tissue-derived the most abundant adult stem cell One of, oligodendroglia, which is prepared, using MSCs had both avoided the oligodendroglia cell preparation using the source ESCs and generate Serious ethics problem and in vivo transplanting tumorigenesis bad clinical risk, also avoid using the source iPSCs less dash forward glue The bad clinical risk of foreign gene transfection and more serious internal transplanting oncogenicity that cell plastid preparation generates, and operate letter It is single, OPCs high income, non-animal derived ingredient.

Detailed description of the invention

Fig. 1 is cellular morphology figure of the P2 for MSCs;

Fig. 2 is P2 for MSCs cell expression nestin aspect graph；

Fig. 3 is P2 for MSCs cell expression A2B5 aspect graph；

Fig. 4 is neural epithelium pre-induced cellular morphology figure；

Fig. 5 is neural epithelium pre-induced cell expression nestin aspect graph；

Fig. 6 is neural epithelium pre-induced cell expression A2B5 aspect graph；

Fig. 7 is cellular morphology figure of the P0 for OPCs;

Fig. 8 is P0 for OPCs cell expression nestin aspect graph；

Fig. 9 is P0 for OPCs cell expression A2B5 aspect graph；

Figure 10 is cellular morphology figure of the P2 for OPCs;

Figure 11 is P2 for OPCs cell expression nestin aspect graph；

Figure 12 is P2 for OPCs cell expression A2B5 aspect graph；

Figure 13 is the statistical chart of the expression of different cultivation stage cell sign albumen；

Figure 14 is different cultivation stage cell yield statistical charts；

Figure 15 is oligodendroglia aspect graph；

Figure 16 is oligodendroglia expression nestin aspect graph；

Figure 17 is oligodendroglia expression A2B5 aspect graph；

Figure 18 is oligodendroglia expression MBP aspect graph.

Specific embodiment

Below in conjunction with specific embodiment, technical scheme in the embodiment of the invention is clearly and completely described.It is real Apply various reagents used in example, machinery is commercially obtained without specified otherwise.

Firstly, to the factory of various products specifically used in the English name and embodiment occurred in the application Family, article No. are briefly described.

OPCs: oligodendrocyte precursor cells, oligodendrocyte precursor cells.

ESCs: embryonic stem cell, embryonic stem cells.

MSCs: mesenchyma stromal cells, mesenchymal stroma cells.

DMEM/F12: being a kind of cell culture medium, and DMEM/F12 article No. used in embodiment is 12400-024, raw Producing producer is GIBCO.

B27: human leucocyte antigen (HLA).

N2: being a kind of cell culture additive, N2 article No. used in embodiment is 17502-048, and manufacturer is GIBCO。

PBS: phosphate buffer.

Non-animal derived ingredient serum replacement: non-animal derived ingredient serum replacement article No. used in embodiment is C08003C, manufacturer are Stemboscience.

0.25% trypsin solution: 0.25% trypsin solution article No. is T6424-1VL used in embodiment, at Producing producer is Biological industries.

Aprotinin solution: Aprotinin solution article No. is A1250000 used in embodiment, is Sigma at producer is produced.

FBS: fetal calf serum, article No. 04-400-1A, manufacturer BI.

T3: triiodothyronine, article No. 709611, manufacturer Sigma-Aldrich.

Sonic hedgehog: Sonic hedgehog, sonic hedgehog article No. used in embodiment is SRP3156, Manufacturer is Sigma.

Noggin: noggin, Noggin article No. used in embodiment is H6416, and manufacturer is Sigma.

Double butyryl c-AMP: double butyryl c-AMP article No.s used in embodiment are D0627, and manufacturer is Sigma.

IGF-1: insulin-like growth factor 1, article No. cyt-216, manufacturer Prospec-Tany.

NT3: neurotrophin 3, article No. cyt-257, manufacturer Prospec-Tany.

Embodiment one: the composition of kit

The oligodendroglia reagent preparation box in the source MSCs includes OPCs serum-free basal medium (OBM), OPCs training Support additive 1(OS1), OPCs cultivate additive 2(OS2) and OPCs culture surface coating buffer (OCB).

Wherein, OPCs serum-free basal medium (OBM) is the DMEM/F12 culture medium comprising following substance:

1 × B27,

1 × N2,

2mM L-Glutamine,

1uM 1,25- dihydroxyvitamin D3 (1,25 (OH) 2D3),

40ng/mL triiodothyronine (3,3', 5-tri-iodo-thyronine, T3),

0.5ug/mL people's epiphysin (Melatonin).

OPCs cultivates additive 1(OS1) be the DMEM/F12 culture medium comprising following substance:

1 ug/mL recombination human basic fibroblast growth factor (rh b-FGF),

1 ug/mL recombinant human epidermal growth factor (rh EGF).

OPCs cultivates additive 2(OS2) be the DMEM/F12 culture medium comprising following substance:

1 ug recombination human platelet derived growth factor AA(rh PDGF-AA),

100ng/mL recombinant human nerve trophic factors 3(rh NT-3).

OPCs culture surface coating buffer (OCB) is the DMEM/F12 culture medium comprising following substance:

100ng/mL recombined human Laminin lens (liminin, LN),

100ng/mL recombined human vitronectin (vitronectin (VN)).

The preparation of embodiment two: OPCs

The reagent that the preparation of OPCs uses, except PBS, digestive juice, freeze protection liquid in addition to, be described in embodiment one The oligodendroglia reagent preparation box in the source MSCs provides.

S1. OS1, OS2, OCB that -20 DEG C or less freeze are thawed overnight under the conditions of 4 DEG C spare;

S2. the OCB in kit is diluted to through DMEM/F12 with 10 × OCB(final concentration of 10%) by every milliliter of packet By 35cm²Culture surface room temperature is coated with culture surface 1 hour, is inhaled and is abandoned coating buffer, is rinsed 3 times with 2-8 DEG C of PBS, is placed in 4 DEG C of preservations No more than 1 week；

S3. people's umbilical cord, amnion, amniotic fluid, placenta, Cord blood, palace film, dental pulp, marrow, skin, tendon, skeletal muscle etc. are organized The P2 in source is for MSCs, by 6000/cm²Inoculation, to contain the MSCs culture medium culture of 10% non-animal derived ingredient serum replacement Converge to 60-70%；

S4. the OBM culture medium containing 2%OS1 is changed, culture to 80-90% converges, and is digested and is harvested with 0.25% trypsin solution Cell is denoted as neural epithelium pre-induced cell；

S5. neural epithelium pre-induced cell is resuspended containing the OBM culture medium of 2%OS1 and 2% OS2, adjustment cell density is extremely 1.5×10⁴/cm², passage when being seeded to OCB coated culture surface culture 60-80% converging in 4-6 days is denoted as P0 for OPCs；

For OPCs, routinely attached cell propagating method passes on S6.P0, digestive juice Accutase^TMDigestive juice (article No. 40506ES60, manufacturer Innovative Cell Technologies, Inc.), inoculum density is 1.5 × 10⁴/cm², Cultivating system is the OBM culture medium containing 1%OS1 and 1% OS2；Culture surface is the coated culture surface of OCB；

S7. harvest P2 can directly prepare preparation for cell, can also be with cryopreservation, and freezing protection liquid using article No. is CELLBANKER-II, manufacturer freeze protection liquid for ZENOAQ's, and freeze-stored cell density is 2 × 10⁶/mL。

Embodiment three: the OPCs external preparation in the source umbilical cord MSCs

3.1 umbilical cord MSCs are separately cultured

With tissue block method originally culture umbilical cord MSCs, cultivating system is MSCs culture medium, that is, contains 10% non-animal derived component blood The DMEM/F12 culture medium of clear substitute, when culture was to 12 days, gently shake culture bottle, suspension remnant tissue block, careful inhale are abandoned Culture supernatant, PBS are washed culture surface 1 time, and 0.25% trypsin solution is added, and room temperature digests 5 minutes, and 1mL Aprotinin is added Solution terminates digestion；400g is centrifuged 5min, abandons supernatant, harvests sediment；With PBS washing 2 times, it is sieved through with 100um Nylon cell Filtrate is collected in filter；400g is centrifuged 5min, abandons supernatant, harvests sedimentation cell, is denoted as P0 for MSCs；P0 is cultivated for MSCs with MSCs Base weight is outstanding, and adjustment cell density is 6000/cm², it is seeded to 175cm²Tissue culture flasks (EasyFlask, NUNC), culture to 70- 80% harvests P1 for MSCs when converging；By 6000/cm of cell density²Continue to pass on, harvests P2 for MSCs.

The source 3.2MSCs OPCs preparation

P2 is resuspended for the MSCs in umbilical cord source, by 6000/cm with MSCs culture medium²It is seeded to 175cm²Tissue culture flasks, Culture converges to 60-70%, changes the OBM culture medium containing 2%OS1, and culture to 80-90% converges, and inhales and abandons culture supernatant, PBS washing training It supports surface 1 time, 0.25% trypsin solution is added, room temperature digests 5 minutes, and 1mL Aprotinin solution is added and terminates digestion；400g Centrifugation, 5min abandon supernatant, harvest sedimentation cell, are denoted as neural epithelium pre-induced cell；

Neural epithelium pre-induced cell is resuspended containing the OBM culture medium of 2%OS1 and 2% OS2, by 1.5 × 10⁴/cm²Inoculation To the coated 175cm of OCB²Tissue culture flasks change liquid every other day, when 60-80% converges, inhale and abandon culture supernatant, PBS washing culture table Accutase is added in face 1 time^TMDigestive juice (article No. 40506ES60, manufacturer Innovative Cell Technologies, Inc.) 5mL, room temperature digest 5 minutes, and 1mL Aprotinin solution is added and terminates digestion；400g is centrifuged 5min, abandons supernatant, harvest precipitating Cell is denoted as P0 for OPCs；

P0 is resuspended for OPCs, by 1.5 × 10 containing the OBM culture medium of 2%OS1 and 2% OS2⁴/cm²It is coated to be seeded to OCB 175cm²Tissue culture flasks, harvest when culture converges to 60-80%, are denoted as P1 for OPCs；P1 continues secondary culture to P2 for OPCs Generation, harvest.

Example IV: P2 is detected for the OPCs differentiation potential in the source MSCs

4.1 oligodendrocyte differentiation cultures

By 5 × 10⁴/ mL is inoculated with P2 and cultivates for OPCs in the coated 24 hole tissue culturing plate of OCB, and every two and half amount changes liquid, Row histology at the 8th day.Oligodendrocyte differentiation culture medium is the DMEM/F12 culture medium containing following ingredient:

2% non-animal derived ingredient serum replacement,

2%FBS,

1 × N2,

40ng/mL T3,

100ng/mL sonic hedgehog,

100 ng/ml Noggin,

10 μM of double butyryl,

100 ng/ml IGF-1,

10ng/NT3。

4.2MSCs, the general Biological Detection of neural epithelium pre-induced cell, OPCs, oligodendroglia

4.2.1 immunohistology detects

When attached cell immunohistology detects, inhales and abandon culture medium, it is solid with 4% paraformaldehyde (PFA) with PBS rinsing 2 times Determine cell 10 minutes, with PBS rinsing 2 times, primary antibody is added dropwise, 4 DEG C overnight；PBS is washed 2 times, and FITC is added dropwise and marks secondary antibody, room temperature is incubated It educates 1 hour；PBS is rinsed 2 times, and 1 μ g/ ml 4', 6- diamidino -2-phenylindone (DAPI) is added dropwise, and is incubated for 5 minutes, PBS rinsing 2 times, microscopy.

Immunohistochemical detection includes: using antibody

Mouse anti-nestin[clone 10C2 (1:400), manufacturer Millipore]；

Mouse anti-A2B5[clone 105 (1:500), manufacturer R&D Systems]；

Rabbit anti-MBP [(1:200), manufacturer Sigma-Aldrich]；

FITC marks secondary antibody: the anti-mouse IgG (1:200) of monkey and goat anti-rabbit igg (1:200) (manufacturer Life Technologies).

4.2.2 cell yield counts

With cell imaging system (Cytell Cell Imaging System, GE Healthcare) row viable count With immunohistology dyeing counting, comprising: P2 is for MSCs, nestin+DAPI+ neural epithelium pre-induced cell quantity, nestin+ The P0 of DAPI+, A2B5+DAPI+ are for OPCs and P2 for OPCs quantity, and the P2 of MBP+, DAPI+ are for OPCs and oligodendroglia number Amount.

Statistical analysis carries out statistical procedures using SPSS17.0.Data with± S indicates that mean value compares using independent Sample t-test, P < 0.05 are statistically significant.

Embodiment five: statistical result

5.1 neural epithelium pre-induced cell culture

The expression statistical result of different cultivation stage cell sign albumen is as shown in figure 13.

As shown in Fig. 1 ~ 3,

P2 extends for MSCs with incubation time to be grown in typical circinate, and most cells are spindle shape, and minority is triangle Shape, polygonal (Fig. 1);

11.87 ± 1.15% cell expresses nestin(Fig. 2)；

7.84 ± 0.56% cell expresses A2B5(Fig. 3)；

As shown in Fig. 4 ~ 6,

After the OBM culture medium culture containing 2%OS1, cell is gradually parallel to each other, and retracts cytoplasm, forms obvious elongate Cell body (Fig. 4)；

95.84 ± 8.61% cell expresses nestin(Fig. 5)；

21.84 ± 5.56% cell expresses A2B5(Fig. 6).

5.2P0 being cultivated for OPCs

As shown in Fig. 7 ~ 9

With OBM culture medium culture neural epithelium pre-induced cell 4-6 days containing 2%OS1 and 2% OS2, part attached cell Obtain ambipolar OPCs form (Fig. 7)；

45.21 ± 4.66% cell expresses nestin(Fig. 8)；

46.22 ± 7.39% cell expresses A2B5(Fig. 9).

5.3P2 being cultivated for OPCs

With the OBM culture medium squamous subculture P0 containing 1%OS1 and 1% OS2 for OPCs to P2 generation, most cells shows are bipolar out Property or three polarity, a few cell show multipolarity form (Figure 10)；

41.83 ± 3.91% cell expresses nestin(Figure 11)；

92.09 ± 11.43% cell expresses A2B5(Figure 12).

5.4OPCs cell yield

Different cultivation stage cell yield statistical results are as shown in figure 14.

1.05×10⁶P2 presses 6000/cm for MSCs²It is inoculated into 175cm²In tissue culture flasks (Easyflask, NUNC), Obtain 5.74 ± 1.48 × 10⁶Neural epithelium pre-induced cell；

Neural epithelium pre-induced cell about presses 1.5 × 10⁴/cm²It is seeded to coated 2 175cm of OCB²Tissue culture flasks In (Easyflask, NUNC), 15.66 ± 3.39 × 10 are obtained⁶P0 is for OPCs；

P0 about presses 1.5 × 10 for OPCs⁴/cm²It is seeded to coated 6 175cm of OCB²Tissue culture flasks (Easyflask, NUNC in), 46.51 ± 8.82 × 10 are obtained⁶P1 then about presses 1.5 × 10 for OPCs⁴/cm²It is seeded to OCB coated 18 175cm²In tissue culture flasks (Easyflask, NUNC), 139.89 ± 36.22 × 10 are obtained⁶P2 is for OPCs.

P2 presses 5 × 10 for OPCs⁴/ mL is inoculated in the coated 24 hole tissue culturing plate of OCB and cultivates 8 days, induces oligodendroglia Cell differentiation.The result shows that culture starts on the 4th day, cell stops proliferation, a large amount of cell deaths, until at the 8th day, Average Survival Cell number is 2.24 ± 0.88 × 104/ hole, and all cell differentiations are at typical oligodendroglia sample form (Figure 15).

Immunohistochemical detection the result shows that, 2.83 ± 0.52% survivaling cells express nestin(Figure 16), 9.95 ± 2.22% survivaling cell expresses A2B5(Figure 17), and 98.31 ± 11.42% survivaling cells express MBP(Figure 18).

Embodiment six: interpretation of result

Mammalian central nervous system damage, aixs cylinder demyelinate and oligodendroglia cell death cause myelin to damage It is the major reason for causing Nerve conduction to lose that caused action potential, which propagates interruption, after wound, missing, and damage is thin after occurring Born of the same parents, which are difficult to regenerate, hinders neurological functional recovery.The past studies have shown that cell transplantation, especially neural precursor transplanting lure The raw myelin of artificial delivery can promote aixs cylinder Remyelination, restore complete action potential conduction and nervous function.Embryonic stem cell, nerve A variety of embryos such as stem cell, adult stem cell can be divided into OPCs, and the cell therapy for myelin loss provides potential cell Source.But the ethical hindrances of embryonic stem cell clinical application and in vivo transplanting oncogenicity are as derived from embryonic stem cells One of the biggest obstacle of OPCs treatment demyelinating disease.Postnatal adult acquisition neural stem cell is extremely difficult, and cell concentration is difficult To guarantee, therefore adult neural stem cell, mostly from aborted fetus, ethical hindrances are bigger.So more researchs are focused on into The OPCs that body multipotential stem cell is external evoked, amplification generates.

MSCs is the multipotential stem cell for being present in Various Tissues, has triploblastica differentiation potential.Except adult bone marrow, fat, Outside the tissues such as skin, can it be separated in the tissue such as fetus at perinatal stage adjunct, including umbilical cord, amnion, amniotic fluid, placenta, Cord blood A large amount of MSCs out.Wherein the MSCs in umbilical cord source is easiest to pilot scale culture and amplification, and cell uniformity is good, and have at Bone, at fat, at cartilage, at endothelial progenitor cells, at differentiation potentials such as neural stem cell, immunogenicity is low, not bright after transplanting Aobvious adverse reaction and oncogenicity.Currently, the country has been set up multiple libraries MSCs, thus MSCs become except candidate stem cell it Outside, it is most suitable for the seed cell for cell therapy, the OPCs preparation system in the exploitation source MSCs is that OPCs transplantation treatment takes off marrow Most important technological problems in sheath disease Industrialization Way.The research of the past focuses mostly on dry thin with embryonic stem cell, induced multi-potent The OPCs induction system of born of the same parents, i.e., using " embryoid body culture-nerve ball-OPCs induction differentiation " three step preparation systems, in recent years Come, there is team to prepare the OPCs in the source MSCs using this system, but because of complex process, is difficult to meet the OPCs mark in the source MSCs Standardization, prepare with scale.

Based on the above situation, applicant develops a kind of kit, prepared by the OPCs industrialization for the source MSCs, is not necessarily to By nerve ball cultivation stage, routinely pass on, simple process, and this kit without the animal sources such as any serum at Point, meet requirement of the clinical research to culture medium.Cell preparation is used for clinical research and clinical application, and cell quantity not only needs Meet the requirement of clinical dosage, it is also necessary to meet the requirement of quality control.The past studies have shown that in addition to embryonic stem cell energy A large amount of induction differentiation generate neural stem cell, then differentiation is induced to generate outside OPCs, are difficult to obtain by nerve ball culture and induction The OPCs that quantity is enough, A2B5 positive rate is high.The P2 in the source MSCs is prepared for OPCs, in addition to A2B5 is expressed using this kit Rate is up to 92.09 ± 11.43%(attached drawing 13), external oligodendroglia induction yield is up to 44.04%(attached drawing 14) outside, it compares In P2 for MSCs, expands multiplying power and be up to 133.23 times (attached drawings 14).By taking this laboratory prepares P2 for the MSCs in umbilical cord source as an example, The umbilical cord of a piece 10cm, P2 is for MSCs yield average out to 1.34 ± 1.10 × 10⁹, external preparation should can harvest 1.79 × 10¹¹ P2 fully meets the industrialization preparation demand for establishing the library OPCs for OPCs.Therefore, this kit be it is a it is easy to use, without blood The animal source components such as clear, the efficient kits for preparing A2B5+OPCs.

Claims

1. a kind of oligodendroglia preparation method in the source MSCs, which comprises the steps of:

S1. it is coated with culture surface 1 hour, is inhaled with the OPCs culture surface coating buffer room temperature for diluting 10 times through DMEM/F12 culture medium Coating buffer is abandoned, 4 DEG C is placed on through PBS rinsing and saves no more than 1 week；

The MSCs in S2.P2 generation presses 6000/cm²Inoculation is trained with the MSCs culture medium containing 10% non-animal derived ingredient serum replacement It supports to 60-70% and converges；

S3. the OPCs serum-free basal medium of the culture additive 1 containing OPCs is changed, culture converges to 80-90%, uses trypsase Solution digestion harvests cell, is denoted as neural epithelium pre-induced cell；

S4. neural epithelium is resuspended with the OPCs serum-free basal medium for cultivating additive 1 and OPCs culture additive 2 containing OPCs Pre-induced cell, adjustment cell density to 1.5 × 10⁴/cm², it is seeded to by the coated culture table of OPCs culture surface coating buffer Face, passage when culture converges to 60-80%, is denoted as P0 for OPCs；

Wherein, the OPCs serum-free basal medium is to contain 1 × B27,1 × N2,2mM L-Glutamine, 1uM 1,25- Dihydroxyvitamin D3,40ng/mL triiodothyronine, the DMEM/F12 culture medium of 0.5ug/mL people's epiphysin；

The OPCs culture additive 1 is containing 1 ug/mL recombination human basic fibroblast growth factor, 1 ug/mL weight The DMEM/F12 culture medium of group hEGF；

The OPCs culture additive 2 is containing 1 ug recombination human platelet derived growth factor AA, 100ng/mL recombined human mind DMEM/F12 culture medium through trophic factors 3；

The OPCs culture surface coating buffer is to connect egg containing 100ng/mL recombined human Laminin lens, 100ng/mL recombined human glass White DMEM/F12 culture medium；

OPCs culture adds in the OPCs serum-free basal medium containing OPCs culture additive 1 and OPCs culture additive 2 It is 2% that the content for adding agent 1, which is the content that 2%, OPCs cultivates additive 2,.

2. a kind of oligodendroglia preparation method in source MSCs according to claim 1, which is characterized in that the step Further include following steps after rapid S4: by P0, for OPCs, routinely attached cell propagating method is passed on, and harvests P2 for cell.

3. a kind of oligodendroglia preparation method in source MSCs according to claim 2, which is characterized in that described normal Advising the inoculum density in attached cell propagating method succeeding generations is 1.5 × 10⁴/cm², cultivating system is that the culture containing 1%OPCs adds Agent 1 and 1% OPCs is added to cultivate the OPCs serum-free basal medium of additive 2；Culture surface is to be coated with through OPCs culture surface The coated culture surface of liquid.

4. a kind of oligodendroglia preparation method in source MSCs according to claim 1, which is characterized in that described MSCs derives from people's umbilical cord, amnion, amniotic fluid, placenta, Cord blood, palace film, dental pulp, marrow, skin, tendon, skeletal muscle.