CN107164325A

CN107164325A - The preparation method and kit of the oligodendroglia in MSCs sources

Info

Publication number: CN107164325A
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: 2017-09-15
Anticipated expiration: 2037-06-28
Also published as: CN107164325B

Abstract

The invention discloses a kind of oligodendroglia reagent preparation box in MSCs sources, including OPCs serum-frees basal medium, OPCs cultures additive 1, OPCs culture additives 2 and OPCs culture surface coating buffers, wherein, the OPCs serum-frees basal medium is to contain B27, N2, L glutamine, 1,25 dihydroxyvitamin D3s, triiodothyronine, the DMEM/F12 culture mediums of people's epiphysin；The OPCs cultures additive 1 is the DMEM/F12 culture mediums containing recombination human basic fibroblast growth factor, recombinant human epidermal growth factor；The OPCs cultures additive 2 is the DMEM/F12 culture mediums containing recombination human platelet derived growth factor AA, recombinant human nerve trophic factors 3；The OPCs culture surfaces coating buffer is the DMEM/F12 culture mediums containing recombined human Laminin lens, recombined human vitronectin.

Description

The preparation method and kit of the oligodendroglia in MSCs sources

Technical field

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

Background technology

A variety of nerve degenerative diseases such as multiple sclerosis, leukaemia, malnutrition, and central lesion Deng that all myelin can be caused to lose, effective treatment means are lacked at present.Oligodendrocyte precursor cells（oligodendrocyte Precursor cells, OPCs）Differentiation produces oligodendroglia formation sheath structure, its exception in central nervous system Demyelinating disease of central nervous system can be caused to become, then cause neure damage and mental disorder, be important therapy target. Oligodendrocyte precursor cells replacement therapy or Regeneration and Repair treatment are maintenance and the aixs cylinder for repairing damage, rebuild aixs cylinder electrical conduction work( The latest developments of energy and most promising treatment means.

The preparation of oligodendroglia of the prior art uses embryonic stem cell（Embryonic stem cells, ESCs）, induced multi-potent stem cell（Induced pluripotent stem cells, iPSCs）Vitro differentiation is obtained.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 iPSCs sources is related to foreign gene transfection and the serious of more serious internal transplanting oncogenicity faces Bed risk.It is right using the animal derived components such as serum and ESCs or iPSCs Differentiation Induction in vitro prepares OPCs complex process The OPCs qualities of the pharmaceutical preparations and clinical safety produce the influence that can not be estimated, while limiting OPCs preparation yields, have impact on clinic Research and industrialization Transformation Potential.

The problem of in correlation technique, effective solution is not yet proposed at present.

The content of the invention

For the above-mentioned technical problem in correlation technique, the present invention proposes a kind of system of the oligodendroglia in MSCs sources Preparation Method and kit, both avoid the serious ethics produced using the oligodendroglia cell preparation that ESCs originates Problem and in vivo the bad clinical risk of transplanting tumorigenesis, the oligodendroglia preparation that it also avoid being originated using iPSCs are produced Foreign gene transfection and more serious internal transplanting oncogenicity bad clinical risk, simple to operate, OPCs high incomes, without dynamic Material resource composition.

To realize above-mentioned technical purpose, the technical proposal of the invention is realized in this way：

A kind of oligodendroglia reagent preparation box in MSCs sources, including OPCs serum-frees basal medium, OPCs culture add Plus agent 1, OPCs culture additives 2 and OPCs culture surface coating buffers, wherein,

The OPCs serum-frees basal medium is to contain B27, N2, Glu, 1,25- dihydroxyvitamin D3, triiodo first Shape gland propylhomoserin, the DMEM/F12 culture mediums of people's epiphysin；

The OPCs cultures additive 1 is to contain recombination human basic fibroblast growth factor, recombinant human epidermal growth factor DMEM/F12 culture mediums；

The OPCs cultures additive 2 is containing recombination human platelet derived growth factor AA, recombinant human nerve trophic factors 3 DMEM/F12 culture mediums；

The OPCs culture surfaces coating buffer is the DMEM/F12 cultures containing recombined human Laminin lens, recombined human vitronectin Base.

Further,

The OPCs serum-frees basal medium contains 1 × B27,1 × N2,2mM Glus, 1uM 1, the dimension life of 25- dihydroxies Plain D3,40ng/mL triiodothyronine, 0.5ug/mL people's epiphysin；

The OPCs cultures additive 1 is containing 1 ug/mL recombination human basic fibroblast growth factors, 1 ug/mL weights The DMEM/F12 of group hEGF；

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

The OPCs culture surfaces coating buffer is to connect egg containing 100ng/mL recombined humans Laminin lens, 100ng/mL recombined human glass White DMEM/F12.

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

S1. it is coated with culture surface 1 hour with OPCs culture surface coating buffers room temperature, coating buffer is abandoned in suction, and 4 are placed in after being rinsed through PBS DEG C preserve be no more than 1 week；

The MSCs in S2.P2 generations presses 6000/cm²Inoculation, to be trained containing the MSCs culture mediums of 10% non-animal derived composition serum replacement Support to 60-70% and converge；

S3. the OPCs serum-free basal mediums that additive 1 is cultivated containing OPCs are changed, culture converges to 80-90%, uses trypsase Solution digestion harvesting, is designated as neural epithelium pre-induced cell；

S4. neural epithelium is resuspended with the OPCs serum-frees basal medium that additive 1 and OPCs culture additives 2 are cultivated 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 buffers Face, culture is passed on when converging to 60-80%, is designated as P0 for OPCs.

Further,

The OPCs culture surfaces coating buffer is to dilute 10 times of OPCs culture surface coating buffers through DMEM/F12；

OPCs culture additive 1 contents are 2% in the OPCs serum-free basal mediums that additive 1 is cultivated containing OPCs；

OPCs cultures add in the OPCs serum-free basal mediums that additive 1 and OPCs culture additives 2 are cultivated containing OPCs Plus the content that the content of agent 1 is 2%, OPCs culture additives 2 is 2%.

Further, also comprise the following steps after the step S4：By P0 for OPCs routinely attached cell propagating methods Passage, P2 is for cell for harvest.

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

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 invention provides the method that oligodendroglia is prepared by source of MSCs, and carry A kind of kit that oligodendroglia is prepared by source of MSCs is supplied.MSCs is tissue-derived most abundant adult stem cell One of, using MSCs prepare oligodendroglia both avoid using ESCs originate oligodendroglia cell preparation and produce Serious ethics problem and in vivo transplanting tumorigenesis bad clinical risk, it also avoid using iPSCs originate less dash forward glue Foreign gene transfection and the bad clinical risk of more serious internal transplanting oncogenicity that cell plastid preparation is produced, and operate letter It is single, OPCs high incomes, non-animal derived composition.

Brief description of the drawings

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

Fig. 2 is that P2 expresses nestin aspect graphs for MSCs cells；

Fig. 3 is that P2 expresses A2B5 aspect graphs for MSCs cells；

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

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

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

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

Fig. 8 is that P0 expresses nestin aspect graphs for OPCs cells；

Fig. 9 is that P0 expresses A2B5 aspect graphs for OPCs cells；

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

Figure 11 is that P2 expresses nestin aspect graphs for OPCs cells；

Figure 12 is that P2 expresses A2B5 aspect graphs for OPCs cells；

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 graphs；

Figure 17 is oligodendroglia expression A2B5 aspect graphs；

Figure 18 is oligodendroglia expression MBP aspect graphs.

Embodiment

The technical scheme in the embodiment of the present invention is clearly and completely described below in conjunction with specific embodiment.It is real The various reagents used in example, machinery are applied commercially to obtain without specified otherwise.

First, to the factory of various products specifically used in the English name and embodiment that occur 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：It is that a kind of DMEM/F12 article No.s used in cell culture medium, embodiment are 12400-024, it is raw It is GIBCO to produce producer.

B27:Human leucocyte antigen (HLA).

N2:It is that a kind of N2 article No.s used in cell culture additive, embodiment are 17502-048, manufacturer is GIBCO。

PBS:Phosphate buffer.

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

0.25% trypsin solution：0.25% trypsin solution article No. used in embodiment is T6424-1VL, into It is Biological industries to produce producer.

Aprotinin solution：Aprotinin solution article No. used in embodiment is A1250000, and into production, producer is Sigma.

FBS：Hyclone, article No. 04-400-1A, manufacturer BI.

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

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

Noggin：Noggin article No.s used in noggin, embodiment are 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：NT3, article No. cyt-257, manufacturer Prospec-Tany.

Embodiment one：The composition of kit

The oligodendroglia reagent preparation box in MSCs sources includes OPCs serum-free basal mediums（OBM）, OPCs culture add Plus agent 1（OS1）, OPCs culture additive 2（OS2）And OPCs culture surface coating buffers（OCB）.

Wherein, OPCs serum-frees basal medium（OBM）For the DMEM/F12 culture mediums comprising following material：

1 × B27,

1 × N2,

2mM Glus,

1uM 1,25 dihydroxyvitamin D3s（1,25(OH)2D3）,

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

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

OPCs cultivates additive 1（OS1）For the DMEM/F12 culture mediums comprising following material：

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

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

OPCs cultivates additive 2（OS2）For the DMEM/F12 culture mediums comprising following material：

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

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

OPCs culture surface coating buffers（OCB）For the DMEM/F12 culture mediums comprising following material：

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

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

Embodiment two：OPCs preparation

The reagent that OPCs preparation is used, except PBS, digestive juice, freeze protection liquid in addition to, be that MSCs described in embodiment one comes The oligodendroglia reagent preparation box in source is provided.

S1. less than -20 DEG C OS1, OS2, OCB frozen are stayed overnight into defrosting under the conditions of 4 DEG C standby;

S2. with 10 × OCB（The OCB in kit is diluted to final concentration of 10% through DMEM/F12）By every milliliter of coating 35cm²Culture surface room temperature coating culture surface 1 hour, coating buffer is abandoned in suction, is rinsed 3 times with 2-8 DEG C of PBS, is placed in 4 DEG C and is preserved not 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 tissue-derived P2 for MSCs, by 6000/cm²Inoculation, with the MSCs medium cultures containing 10% non-animal derived composition serum replacement extremely 60-70% converges；

S4. the OBM culture mediums containing 2%OS1 are changed, culture to 80-90% converges, and harvesting is digested with 0.25% trypsin solution, It is designated as neural epithelium pre-induced cell；

S5. to be resuspended neural epithelium pre-induced cell containing 2%OS1 and 2% OS2 OBM culture mediums, adjustment cell density to 1.5 × 10⁴/cm², it is seeded to when the coated 4-6 days 60-80% of culture surface culture of OCB converge and passes on, is designated as P0 for OPCs；

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

S7. harvest P2 is for cell, can direct preparation of preparation, can also cryopreservation, freeze and protect the liquid to be using article No. CELLBANKER-II, manufacturer freezes protection liquid for ZENOAQ's, and freeze-stored cell density is 2 × 10⁶/mL。

Embodiment three：The OPCs external preparations in umbilical cord MSCs sources

3.1 umbilical cord MSCs are separately cultured

With tissue block method original cuiture umbilical cord MSCs, cultivating system is MSCs culture mediums, i.e., replaced containing 10% non-animal derived composition serum Dai Pin DMEM/F12 culture mediums, when cultivating to 12 days, gently concussion and cultivate bottle, suspension remnant tissue block, carefully inhale and abandon culture Supernatant, PBS washings culture surface 1 time, adds 0.25% trypsin solution, and room temperature digests 5 minutes, adds 1mL Aprotinin solution Terminate digestion；400g centrifuges 5min, abandons supernatant, harvests sediment；Washed 2 times with PBS, filter is sieved through with 100um Nylon cells, received Collect filtrate；400g centrifuges 5min, abandons supernatant, harvests sedimentation cell, is designated as P0 for MSCs；P0 is for MSCs with MSCs culture medium weights Outstanding, adjustment cell density is 6000/cm², it is seeded to 175cm²Tissue culture flasks（EasyFlask, NUNC）, culture to 70-80% P1 is harvested when converging for MSCs；By 6000/cm of cell density²Continue to pass on, P2 is for MSCs for harvest.

It is prepared by 3.2MSCs sources OPCs

The MSCs that P2 originates for umbilical cord is resuspended with MSCs culture mediums, by 6000/cm²It is seeded to 175cm²Tissue culture flasks, training Support to 60-70% and converge, change the OBM culture mediums containing 2%OS1, culture to 80-90% converges, culture supernatant, PBS washing cultures are abandoned in suction Surface 1 time, adds 0.25% trypsin solution, and room temperature digests 5 minutes, adds 1mL Aprotinins solution and terminates digestion；400g from The heart, 5min abandons supernatant, harvests sedimentation cell, is designated as neural epithelium pre-induced cell；

So that neural epithelium pre-induced cell is resuspended containing 2%OS1 and 2% OS2 OBM culture mediums, by 1.5 × 10⁴/cm²It is seeded to The coated 175cm of OCB²Tissue culture flasks, change liquid every other day, when 60-80% converges, and culture supernatant, PBS washing culture surfaces 1 are abandoned in suction It is secondary, add Accutase^TMDigestive juice（Article No. 40506ES60, manufacturer Innovative Cell Technologies, Inc.）5mL, room temperature digests 5 minutes, adds 1mL Aprotinins solution and terminates digestion；400g centrifuges 5min, abandons supernatant, harvest precipitation Cell, is designated as P0 for OPCs；

So that P0 is resuspended for OPCs containing 2%OS1 and 2% OS2 OBM culture mediums, by 1.5 × 10⁴/cm²It is seeded to OCB coated 175cm²Tissue culture flasks, culture is harvested when converging to 60-80%, is designated as P1 for OPCs；P1 continues Secondary Culture to P2 for OPCs Generation, harvest.

Example IV：P2 is detected for the OPCs differentiation potentials that MSCs originates

4.1 oligodendrocyte differentiation cultures

By 5 × 10⁴/ mL is inoculated with P2 and cultivated for OPCs in the coated 24 hole tissue culturing plates of OCB, and every two and half amount changes liquid, the 8th day When row histology.Oligodendrocyte differentiation culture medium is the DMEM/F12 culture mediums containing following composition：

2% non-animal derived composition 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, neural epithelium pre-induced cell, OPCs, the general Biological Detection of oligodendroglia

4.2.1 immunohistology is detected

When attached cell immunohistology is detected, culture medium is abandoned in suction, is rinsed 2 times with PBS, with 4% paraformaldehyde（PFA）It is fixed thin Born of the same parents 10 minutes, are rinsed 2 times with PBS, primary antibody are added dropwise, 4 DEG C overnight；PBS is washed 2 times, and FITC mark secondary antibodies, incubation at room temperature 1 is added dropwise Hour；PBS is rinsed 2 times, and 1 μ g/ ml 4', 6- diamidino -2-phenylindones are added dropwise（DAPI）, it is incubated 5 minutes, PBS rinsings 2 It is secondary, microscopy.

Immunohistochemical detection is included 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 of monkey:200) with goat anti-rabbit igg (1:200)（Manufacturer Life Technologies）.

4.2.2 cell yield is counted

With cell imaging system（Cytell Cell Imaging System, GE Healthcare）Row viable count and exempt from Epidemic disease histological stain is counted, including：P2 is for MSCs, nestin+DAPI+ neural epithelium pre-induced cell quantity, nestin+DAPI +, A2B5+DAPI+ P0 for OPCs and P2 for OPCs quantity, MBP+, DAPI+ P2 are for OPCs and oligodendrocyte numbers.

Statistical analysis carries out statistical procedures using SPSS17.0.Data with± S represents that average ratio is relatively using independent Sample t-test, P<0.05 is 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 for MSCs with incubation time extension in the growth of typical swirling, most cells are spindle shape, and minority is triangle, many It is angular（Fig. 1）;

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

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

As shown in Fig. 4 ~ 6,

After the OBM medium cultures containing 2%OS1, cell is gradually parallel to each other, retraction cytoplasm, and it is thin that formation is substantially elongated Cell space（Fig. 4）；

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

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

5.2P0 cultivated for OPCs

As shown in Fig. 7 ~ 9

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

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

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

5.3P2 cultivated for OPCs

With the OBM culture medium squamous subculture P0 containing 1%OS1 and 1% OS2 for OPCs to P2 generations, most cells shows go out bipolarity or Three polarity, a few cell shows multipolarity form（Figure 10）；

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

92.09 ± 11.43% cell expression 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 175cm²Tissue culture flasks（Easyflask, NUNC）In, 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 （Easyflask, NUNC）In, obtain 15.66 ± 3.39 × 10⁶P0 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, obtain 46.51 ± 8.82 × 10⁶P1 then about presses 1.5 × 10 for OPCs⁴/cm²It is seeded to coated 18 of OCB 175cm²Tissue culture flasks（Easyflask, NUNC）In, obtain 139.89 ± 36.22 × 10⁶P2 is for OPCs.

P2 presses 5 × 10 for OPCs⁴/ mL is inoculated in the coated 24 hole tissue culturing plates of OCB and cultivated 8 days, induces oligodendroglia Cell differentiation.As a result show, culture starts on the 4th day, cell stops breeding, a large amount of cell deaths, during to the 8th day, Average Survival Cell number is 2.24 ± 0.88 × 104/ hole, and all cell differentiations are into typical oligodendroglia sample form（Figure 15）.

Immunohistochemical detection 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 expression MBP（Figure 18）.

Embodiment six：Interpretation of result

Mammalian central nervous system is damaged, and aixs cylinder demyelinate and oligodendroglia cell death cause myelin damage, lacked It is the major reason for causing Nerve conduction to lose that action potential caused by after mistake, which propagates interruption, and cell is difficult to after damage occurs Regeneration hinders neurological functional recovery.The research of the past shows that cell transplantation, particularly neural precursor transplanting induction are produced Myelin can promote aixs cylinder Remyelination, recover complete action potential conduction and nervous function.Embryonic stem cell, NSC OPCs can be divided into etc. a variety of embryos, adult stem cell, the cell therapy lost for myelin provides potential cell derived.But It is that the OPCs treatments that the ethical hindrances of embryonic stem cell clinical practice and in vivo transplanting oncogenicity turn into derived from embryonic stem cells are de- One of biggest obstacle of myelin disease.Postnatal adult acquisition NSC is extremely difficult, and cell concentration is difficult to ensure that, therefore Adult neural stem cell multi-source is in aborted fetus, and ethical hindrances are bigger.So more researchs focus on many abilities of adult carefully The OPCs that born of the same parents are external evoked, amplification is produced.

MSCs is the multipotential stem cell for being present in Various Tissues, with triploblastica differentiation potential.Except adult bone marrow, fat, The tissue such as skin is outer, fetus at perinatal stage adjunct, including can be separated in the tissue such as umbilical cord, amnion, amniotic fluid, placenta, Cord blood Go out substantial amounts of MSCs.The MSCs in wherein umbilical cord source is easiest to pilot scale culture and amplification, and cell uniformity is good, and has into Bone, into fat, into cartilage, into endothelial progenitor cells, into differentiation potentials such as NSCs, immunogenicity is low, not bright after transplanting Aobvious adverse reaction and oncogenicity.At present, the country has been set up multiple MSCs storehouses, thus MSCs turn into except candidate stem cell it Outside, it is most suitable for the seed cell for cell therapy, the OPCs preparation systems in exploitation MSCs sources are that OPCs transplantation treatments take off marrow Most important technological problemses 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 systems of born of the same parents, i.e., using " embryoid body culture-nerve ball-OPCs inductions are broken up " three step preparation systems, in recent years Come, there is team to prepare the OPCs in MSCs sources using this system, but because of complex process, it is difficult to meet the OPCs marks in MSCs sources Standardization, prepare with scale.

Based on above-mentioned situation, applicant develops a kind of kit, is prepared for the OPCs industrialization that MSCs originates, without By neural ball cultivation stage, routinely pass on, technique is simple, and this kit without the animal sources such as any serum into Point, meet requirement of the clinical research to culture medium.Cell preparation is used for clinical research and clinical practice, and its cell quantity is not only needed Meet the requirement of clinical dosage, in addition it is also necessary to meet the requirement of quality control.The research of the past shows, except embryonic stem cell energy A large amount of induction differentiation produce NSC, then induce differentiation to produce outside OPCs, it is difficult to be obtained by neural ball culture and induction Quantity it is enough, the OPCs that A2B5 positive rates are high.The P2 in MSCs sources is prepared for OPCs using this kit, except A2B5 expression Rate is up to 92.09 ± 11.43%（Accompanying drawing 13）, external oligodendroglia induction yield is up to 44.04%（Accompanying drawing 14）Outside, compare In P2 for MSCs, amplification multiplying power is up to 133.23 times（Accompanying drawing 14）.Exemplified by the MSCs that P2 originates for umbilical cord being prepared by this laboratory, Piece 10cm umbilical cord, 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 setting up OPCs storehouses for OPCs.Therefore, this kit be it is a it is easy to use, without blood Clear wait animal derived components, the efficient kit for preparing A2B5+OPCs.

Claims

1. a kind of oligodendroglia reagent preparation box in MSCs sources, it is characterised in that cultivated including OPCs serum-frees basis Base, OPCs cultures additive 1, OPCs culture additives 2 and OPCs culture surface coating buffers, wherein,

2. a kind of oligodendroglia reagent preparation box in MSCs sources according to claim 1, it is characterised in that

3. a kind of oligodendroglia preparation method in MSCs sources, it is characterised in that comprise the following steps：

4. a kind of oligodendroglia preparation method in MSCs sources according to claim 3, it is characterised in that

5. a kind of oligodendroglia preparation method in MSCs sources according to claim 3, it is characterised in that the step Also comprise the following steps after rapid S4：By P0, for OPCs, routinely attached cell propagating method is passed on, and P2 is for cell for harvest.

6. the oligodendroglia preparation method in a kind of MSCs sources according to claim 5, it is characterised in that described normal It is 1.5 × 10 to advise the inoculum density in attached cell propagating method succeeding generations⁴/cm², cultivating system is that the culture containing 1%OPCs adds Plus the OPCs of agent 1 and 1% cultivates the OPCs serum-free basal mediums of additive 2；Culture surface is to be coated with through OPCs culture surfaces The coated culture surface of liquid.

7. the oligodendroglia preparation method in a kind of MSCs sources according to claim 3, it is characterised 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.