CN107232182A

CN107232182A - A kind of mesenchymal stem cells MSCs cell cryopreservation agent

Info

Publication number: CN107232182A
Application number: CN201710528049.8A
Authority: CN
Inventors: 陈印平
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-06-30
Filing date: 2017-06-30
Publication date: 2017-10-10

Abstract

The invention provides a kind of mesenchymal stem cells MSCs cell cryopreservation agent, it contains 0.2%w/v sucrose, 0.5%w/v low-density lipoproteins, 3%w/v trehalose, 2%w/v lecithin, polypeptide 100ppmw/v, bFGF 1.5%w/v, vitamin E 1%w/v, glycerine 5%v/v are finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml.Agent is frozen in the cell of the present invention; glycerine and trehalose and vitamin E and polypeptide, which have, clearly resists damage of the external injury to cell; polypeptide also has damage of the ice crystal to cell when protecting cells from freezing, and remaining several component is necessary for maintaining the existence of specific MSCs cells.The cell cryopreservation agent that the present invention is provided is used to freeze culture cell, in particular improves the survival rate after freeze-stored cell recovery.With preferable application prospect.

Description

A kind of mesenchymal stem cells MSCs cell cryopreservation agent

Technical field

The present invention relates to histocyte culture technique field, and in particular to a kind of mesenchymal stem cells MSCs cell freezes Deposit agent and its preparation method and application.

Background technology

In marrow except containing can differentiation and development into various haemocytes candidate stem cell in addition to, also containing producing non-hematopoiesis group The mescenchymal stem cell (MSCs) knitted, because it is easier adherent and forms fibroblast-like clone, therefore the document having also MSC, referred to as attached cell (fibroblastic colony units-forming (CFU-F).Due to their support structures from marrow, and And can as trophoderm hematopoiesis support stem cell growth, therefore also someone is called marrow stromal cell.

There is MSCs multi-lineage potential to be verified by internal and external test.Friedenstem first confirmed that MSG, With the ability for being divided into bone, cartilage, fibr tissue.Pittenger in 1999 etc. is separated from the ilium sample of bone marrow of people MSCs has been arrived, in vitro under the induction of different differentiation conditions, Gegenbaur's cell, cartilage cell or fat cell can be formed, and The later cell of clone has similar differentiation characteristic, and it is pluripotent stem cell fully to prove bone marrow MSCs.But, Pittenger et al. experiment can not still confirm the MSG being separated to, whether be Committed progenitor cells mixture, in marrow whether Really there is the single stem cell with multi-lineage potential.Halleu etc. by the mesenchymal cell being separated to dilute after with low close Degree inoculation, the clone as a result grown by individual cells can continuous amplification cultivation generation more than 20, and can be divided into skeletonization in vitro Cell, chondroblast, lipoblast, do thin so as to confirm and contain to have in the mesenchymal cell separated in marrow Born of the same parents' characteristic is the individual cells of height self-renewal capacity and polyphyly differentiation potential.Anita etc. is by using marrow stromal cell Clone's culture, the human bone marrow substrate cell clone of analysis 185 non-infinite propagation is divided into skeletonization, into cartilage, into fatty thin The ability of born of the same parents, it was confirmed that MSCs vitro differentiation model:The cell that these three cell lines can be divided into is the mesenchyma ancestral of early stage Cell, the orderly loss of its differentiation potential causes the direction of differentiation to gradually reduce.On MSG, the report of multipotency over nearly 20 years Road is a lot, and this kind of in vitro study experiment is to employ a variety of derivants for different differentiation directions, and MSCs is made respectively Break up to different thesocytes such as skeletonization, into cartilage, into fat, sarcoblast.

MSCs has convenient material drawing, and culture in vitro can keep its multi-lineage potential, and genetic background is stable, implants It is preferable seed cell in organizational project without immunological rejection, it is easy to the features such as clinical practice.Although multidirectional point of MSCs Changing potential is widely confirmed, but its in vivo studies is only limitted to be repaired the tissue of itself with toy MSCs and lacked Damage.In order to which MSCs is used for into big animal and the primate close with the mankind, Fu Wenyu etc. notes people's bone marrow MSCs of culture Enter nude mice by subcutaneous, after one month injection site observed people MSCs differentiation bone, cartilage, fat, skeletal muscle, tendon forceps sample group The Various Tissues type such as knit.Tippi etc. establishes people's tire sheep model of allogene, by people MSG, is implanted in tire sheep, it was observed that People MSCs was survived in tire sheep up to 13 months, and site specific be divided into fat, cartilage, myocyte etc., further MSCs still has polyphyly differentiation potential after confirming transplanting, it was further observed that in the damage location of people's tire sheep model, MSCs points of people The cell concentration increase of change, thus it is speculated that be probably implantation MSCs be induced to expand and participate under this damaging conditions tissue repair or Person's regenerative response.The research is those the ostosis defects that can be just diagnosed in uterus, the disease such as muscular dystrophy Clinical treatment is laid a good foundation.

MSCs is easier to separate from the marrow of patient, in vitro after many times of amplifications, carries out a variety of purposes Operation, then again defeated time in patient body, freezing is standby the need for the cell for preparing therebetween is inevitable.At present, do The frozen stock solution that cell cryopreservation is used has two classes containing serum and serum-free, and because serum has, complicated component, quality be unstable, valency The shortcomings of lattice are expensive, serum-free freezes turns into development trend with resuscitation technique.Cord blood stem cell relies primarily on anti frozen liquid two The protection of methyl sulfoxide (DMSO) and serum.Commonly used cell-protecting also has HES 0ES), polyethylene glycol (PEG) Deng.But, DMSO can produce toxic action to cell, and irreversible damage is caused to the stem cell frozen.To obtain source side Just MSCs stem cells, it is the urgent demand in this area to carry out effective MSCs stem cell cryopreservings method, sets up a kind of long-term low Temperature preserves the method for MSCs stem cells for promoting grinding for MSCs stem cells to make internal disorder or usurp and using being significant.

The content of the invention

The technical problem to be solved in the present invention is to provide a kind of mesenchymal stem cells MSCs cell cryopreservation agent and accordingly Preparation method and application method.

Technical scheme is as follows：

A kind of mesenchymal stem cells MSCs cell cryopreservation agent, it is characterised in that be made up of following each component：0.2%w/v sugarcanes Sugar, 0.5%w/v low-density lipoproteins, 3%w/v trehalose, 2%w/v lecithin, polypeptide 100ppm w/v, bFGF 1.5% W/v, vitamin E 1%w/v, glycerine 5%v/v are finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml, the sequence such as SEQ ID NO of the polypeptide：Shown in 1-2 is any.The polypeptide is directed to by inventor by specificity The substantial amounts of library immunoscreening for the early stage that MSCs cells are carried out is obtained, and the polypeptide, which has, protects MSCs cells from the work(of damage Energy.Its title is respectively MSCs-1 (SEKTWFVWYSWRKQGCIEDSRPTYHYC), MSCs-2 (KAREWEPDRHMTHWIPGFHHMGCSYH)。

In the frozen stock solution of the cell of the present invention, glycerine and trehalose and vitamin E and polypeptide, which have, clearly to be resisted Damage of the external injury to cell, particularly polypeptide, the also damage with ice crystal when protecting cells from freezing to cell, its Component is necessary for maintaining the existence of specific MSCs cells in complementary set.

Present invention also offers a kind of preparation method of the frozen stock solution of cell, will each component mixing shake up.

Present invention also offers a kind of mesenchymal stem cells MSCs cryopreservation methods, comprise the following steps:

A. prepared by frozen stock solution:Described cells frozen storing liquid is prepared, by each component according to conventional operating method by each component It can be obtained according to the mixing of corresponding ratio；

B. prepared by cell suspension:Culture grows into one bottle of the MSCs cells of individual layer, and 0.25% trypsin solution digests 4 points Clock, abandons trypsin solution, adds DMEM nutrient solution 15ml, and gently being blown and beaten with suction pipe makes cell uniform, centrifuges 4000 revs/min, abandons supernatant, Step A frozen stock solution 2ml are added, is mixed, inserts in sterile cryopreservation tube；

C. freeze:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 DEG C is placed into and freezes 3h, finally moves into liquid nitrogen and preserves；

D. cell recovery:Take out cryopreservation tube and be quickly placed into 37 DEG C of water-baths, concussion is melted completely up to cell suspension, Ran Houyong 5 times of DMEM liquid dilutions, 1000r/min centrifugation 5min, centrifugation removes supernatant, repeated 3 times.The cell suspended concentration is 10⁸Cell/ml-10¹⁰Cell/ml.

Beneficial effects of the present invention:

The cells frozen storing liquid of the present invention, recovery cell survival rate relatively uses regular growth frozen stock solution up to more than 98.5% Recovery survival rate be obviously improved, there is no the loss of cell.

The MSCs stem cell cryopreserving liquids of the present invention can preserve MSCs stem cells for a long time, and cytoactive does not change, and protect Activity of cell biology is demonstrate,proved.

Nerve cell cryopreservation methods of the present invention, operation is simple and feasible, affordable, with preferable practical value.

Embodiment

Embodiment 1

It is separately added into 0.2%w/v sucrose, 0.5%w/v low-density lipoproteins, 3%w/v trehalose, 2%w/v lecithins Fat, polypeptide 100ppm w/v, bFGF 1.5%w/v, vitamin E 1%w/v, glycerine 5%v/v finally use DMEM culture mediums and tire Cow's serum is according to 1:1 ratio is settled to 100ml.The wherein sequence of polypeptide such as SEKTWFVWYSWRKQGCIEDSRPTYHYC institutes Show.

Embodiment 2

It is separately added into 0.2%w/v sucrose, 0.5%w/v low-density lipoproteins, 3%w/v trehalose, 2%w/v lecithins Fat, polypeptide 100ppm w/v, bFGF 1.5%w/v, vitamin E 1%w/v, glycerine 5%v/v finally use DMEM culture mediums and tire Cow's serum is according to 1:1 ratio is settled to 100ml.The wherein sequence of polypeptide such as KAREWEPDRHMTHWIPGFHHMGCSYH institutes Show.

Comparative Example I

70ml MEM cell culture fluids, 20ml calf serum, I0ml dimethyl sulfoxide (DMSO) is measured respectively, is mixed to prepare Regular growth frozen stock solution.

Comparative Example I I

By dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 1%w/v trehalose, 1%w/v lecithin, BFGF 1%w/v, vitamin E 1%w/v, finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml。

Comparative Example I II

By dimethyl sulfoxide (DMSO) 5%w/v, 0.5%w/v low-density lipoproteins, 1%w/v trehalose, 1%w/v lecithin, Polypeptide 1%w/v, bFGF 1%w/v, vitamin E 1%w/v, finally with DMEM culture mediums and hyclone according to 1:1 ratio It is settled to 100ml.

The compliance test result of the frozen stock solution of embodiment 3

Cells frozen storing liquid prepared by above example 1-2 and Comparative Example I-III, carries out cell by the following method respectively Freeze and recovering experiment.

Cell cryopreservation process:

Culture grows into the human leukemia stem cell of individual layer, its cell density about 6*10⁹Individual/ml, adds pH7.0 PBS Wash cell surface once.

Cell is digested 4 minutes with 0.25% trypsin solution, trypsin solution is abandoned, DMEM nutrient solution 15ml is added, uses suction pipe Gently piping and druming makes cell uniform, centrifuges 4000 revs/min, abandons supernatant, adds embodiment 1-3 and the frozen stock solution prepared by comparative example 1 2ml, is mixed, and inserts in sterile cryopreservation tube；

Freezing:Cryopreservation tube is first frozen into 30min at 4 DEG C, -30 DEG C is then placed in and freezes lh, -80 DEG C is placed into and freezes 3h, finally moves into liquid nitrogen and preserves；Freeze respectively 1 week, 4 months, 8 months.Every group of 3 repetitions.

Cell recovery:Take out cryopreservation tube and be quickly placed into 37 DEG C of water-baths, concussion until cell suspension melts completely, then with 5 Times DMEM liquid dilution, 1000r/min centrifugation 5min, centrifugation removes supernatant, repeated 3 times, calculates cell survival rate.As a result such as Under：

The freeze-stored cell of 8 months is taken, cell differentiation culture is carried out, wherein described MSCs cells can be normally carried out point Change, differentiation efficiency reaches 95.3%, and Comparative Example I-III takes out cell its differentiation rate can only achieve 58.4%, 60.2%, 80.5%, this sufficiently illustrates that cytoactive receives very big influence.

The specific detection of embodiment 4

By the culture medium of embodiment 1, respectively for other several stem cells：Candidate stem cell, NSC, fat is dry Cell carries out carrying out refrigeration test using the training method of embodiment 3, freezes experiment by 8 months, it is found that after 8 months This several cell survival rate be between 80%-90% survival rate, illustrate, refrigerant of the invention can be with specific Cultivate MSCs.

Above-described embodiment is not limited for the present invention preferably embodiment, but embodiments of the present invention by embodiment System, other any Spirit Essences and the change made under principle, modifications without departing from the present invention, combines, substitutes, simplifying and should be Equivalence replacement mode, is included within protection scope of the present invention.

Sequence table

The ＞ Chen Yin of ＜ 110 are put down

A kind of mesenchymal stem cells MSCs cell cryopreservation agent of the ＞ of ＜ 120

〈160〉2

〈210〉1

〈211〉27

〈212〉PRT

The ＞ artificial sequences of ＜ 213

〈400〉MSCs-1

SEKTWFVWYSWRKQGCIEDSRPTYHYC

〈211〉26

〈212〉PRT

The ＞ artificial sequences of ＜ 213

〈400〉MSCs-2

KAREWEPDRHMTHWIPGFHHMGCSYH

Claims

1. a kind of cell cryopreservation agent, it is characterised in that:It can specific protection MSCs.

2. a kind of cell cryopreservation agent, is made up of following each component：0.2% w/v sucrose, 0.5% w/v low-density lipoproteins, 3% W/v trehalose, 2% w/v lecithin, polypeptide 100ppm w/v, bFGF 1.5% w/v, the w/v of vitamin E 1%, glycerine 5% V/v is finally with DMEM culture mediums and hyclone according to 1:1 ratio is settled to 100ml, the sequence such as SEQ ID of the polypeptide NO：Shown in 1-2 is any.

3. the preparation method of the cells frozen storing liquid described in claim any one of 1-2, it is characterised in that including mixing the formula The component of ratio, obtains described freezing agent.

4. the cells frozen storing liquid described in claim any one of 1-2 is deposited after raising freezes mesenchymal stem cells MSCs cell recovery Application in motility rate.