CN109810943A - One boar muscle-derived mescenchymal stem cell isolation medium and isolated culture method - Google Patents

Abstract

The present invention relates to a boar muscle-derived mescenchymal stem cell isolation medium and isolated culture methods, belong to stem cells technology field, the culture medium is basic culture medium with the α-MEM culture medium containing fetal calf serum, also contains fibroblast growth factor 4, niacinamide and platelet derived growth factor in the basal medium.When progress cell is separately cultured, first by collagenase digestion, mescenchymal stem cell separation is aseptically carried out from pig muscular tissue, then the pig muscle-derived mescenchymal stem cell isolation medium is utilized to carry out continuous purification culture.The present invention by adding active constituent in the medium, make its lower cost and it is relatively simple easy on the basis of obtain pig muscle-derived mescenchymal stem cell, while improving the ability of its division growth ability and secondary culture.

Description

One boar muscle-derived mescenchymal stem cell isolation medium and isolated culture method

Technical field

The invention belongs to stem cells technology fields, and in particular, to a boar muscle-derived mescenchymal stem cell is separately cultured Base and isolated culture method.

Background technique

Tissue mescenchymal stem cell is the stem cell that a group has multi-lineage potential, at present from marrow, fat and bone Mescenchymal stem cell is largely separated in the tissue such as film.Containing there are many stem cell in muscle, some cells have to fat cell, Osteoblast and myoblast differentiation characteristic.

Currently, more to the isolated culture method of tissue mescenchymal stem cell, the characteristics of from method in view of, can generally divide Be two kinds: one is adhere-wall culture screening methods, and one is fluidic cell separating methods.Wherein adhere-wall culture screening method is more common, It is easier to adherent feature by thousand cell of mesenchyma compared with remaining cell mixing, reaches a point cellifugal purpose.Specific to Experimental implementation, is further divided into two classes, and one kind is enzyme digestion, and another is the direct adherent method of tissue, the main distinction be When obtaining primary cell, whether tissue passes through the digestion of enzyme.Enzyme for vitellophag mainly has clostridiopetidase A I, clostridiopetidase A at present IV, hyaluronidase and pancreatin.Fluidic cell sorting rule mainly uses cell size difference not of the same race or surface marker not Same method achievees the purpose that sort mescenchymal stem cell.In contrast, this method for separating primary cells number compared with It is few, and the number for being used to detect the surface markers of the mescenchymal stem cell in cultivating is more.

It is of great significance on basic scientific research and production practices for being separately cultured for mescenchymal stem cell.Streaming Cell sorting methods carry out the method high operation requirements of mescenchymal stem cell separation, and complex steps, cost is also higher, therefore adherent training Feeding screening method is still the more common method in current most of laboratories.When stationary culture mainly utilizes adherent between cell Between and adherent ability difference and mescenchymal stem cell the features such as division growth ability is stronger compared with remaining mature cell and Achieve the purpose that be separately cultured.The tissue mescenchymal stem cell that separation obtains is carried out in secondary culture, at present passage number Limitation be still conditionality factor in experimental study.

Summary of the invention

In order to solve deficiency in the prior art, the purpose of the present invention is to provide a boar muscle-derived mescenchymal stem cells Isolation medium, and further provide for its isolated culture method.The present invention by adding active constituent in the medium, make its Lower cost and it is relatively simple it is easy on the basis of obtain pig muscle-derived mescenchymal stem cell, while improving its division growth ability With the ability of secondary culture.

To achieve the goals above, the present invention use the specific scheme is that

One boar muscle-derived mescenchymal stem cell isolation medium is basic culture medium with the α-MEM culture medium containing fetal calf serum, Also contain fibroblast growth factor 4, niacinamide and platelet derived growth factor in the basal medium.

It is advanced optimized as to above scheme, the pig muscle-derived mescenchymal stem cell isolation medium, to contain body Fraction is that the α-MEM of 20% fetal calf serum is basic culture medium, also contains final concentration of 0.5-2ng/ in the basal medium The blood platelet of the fibroblast growth factor 4 of mL, the niacinamide of final concentration of 100-500 μm of ol/L and final concentration of 3-10ng/mL Source property growth factor.

As the further optimization to above scheme, the pig muscle-derived mescenchymal stem cell isolation medium is described The volume fraction of fetal calf serum is 20% in basal medium, the concentration of fibroblast growth factor 4 is 1ng/mL, niacinamide it is dense Degree is 300 μm of ol/L and the concentration of platelet derived growth factor is 5ng/mL.

It is dry using the mescenchymal stem cell isolation medium progress pig muscle-derived mesenchyma that the present invention also provides a kind of It is dry aseptically to carry out mesenchyma first by collagenase digestion from pig muscular tissue for the method that cell is separately cultured Cell separation, then carries out continuous purification culture using the pig muscle-derived mescenchymal stem cell isolation medium.

As advanced optimizing for the method being separately cultured to above-mentioned pig muscle-derived mescenchymal stem cell, the behaviour of the method Steps are as follows for work:

Step 1: pig longissimus dorsi muscle musculature is cut into small pieces, it is ground into paste homogenate, collected with PBS and washs acquisition is thin Born of the same parents' suspension；Cell suspension is added in the Tissue Culture Flask containing clostridiopetidase A, cultivates 1.5h under the conditions of 37 DEG C, in incubation Repeatedly concussion mixes, and obtains digestive juice；The quality volume fraction of clostridiopetidase A is 2% in the Tissue Culture Flask；

Step 2: mescenchymal stem cell isolation medium is prepared, it is spare；

Step 3: the culture medium that step 2 preparation will be added after digestive juice centrifuge washing obtained by step 1, blows and beats cell with suction pipe, Single cell suspension is made, is then inoculated in the Tissue Culture Flask for being covered with gelatin, 37 DEG C, 5 %CO₂Under the conditions of saturated humidity After cultivating 2h, whole media transfers are continued into adhere-wall culture 2h into another Tissue Culture Flask with suction pipe, while former thin It additionally incorporates fresh culture medium in born of the same parents' culture bottle to continue to cultivate, such continuous purification 3 times or more；Take repeatedly purify it is adherent thin Born of the same parents merge culture, when cell is merged up to 60 % with the pancreatin had digestive transfer culture culture of 0 .25 %.

The utility model has the advantages that

It include activity relevant to cell division proliferation in pig muscle-derived mescenchymal stem cell isolation medium of the present invention Ingredient, the pig muscle-derived mescenchymal stem cell isolated culture method established based on this have step simple, operability By force, and experimentation cost can greatly be reduced, while the proliferation of cell can be effectively facilitated, improve muscle-derived mescenchymal stem cell Purity is cultivated, and the secondary culture number of mescenchymal stem cell can be effectively improved.

Specific embodiment

Below in conjunction with the embodiment of the present invention, technical solution of the present invention is clearly and completely described.

Embodiment (1) material and method

A) test material:

Pig longissimus dorsi muscle muscle.

B) mescenchymal stem cell is separately cultured test

Take pig longissimus dorsi muscle muscle under aseptic condition, test material be cut into small pieces with eye scissors, be put into tissue grinder into Row is ground to paste homogenate, collects cell suspension with PBS, and wash musculature sufficiently with PBS to remove haemocyte.It is put into In Tissue Culture Flask containing 2% clostridiopetidase A, is cultivated 1.5 hours under the conditions of 37 DEG C, during which need to repeatedly shake mixing.Centrifuge washing After be added 5 ml cells be separately cultured liquid suction pipe piping and druming cell for several times, single cell suspension is made, is inoculated in and is covered with the thin of gelatin Born of the same parents' culture bottle is cultivated.Cell is separately cultured liquid system and uses the α-MEM containing 20% fetal calf serum as basic culture medium, together When culture medium in add fibroblast growth factor 4(FGF4) (final concentration 1ng/mL), niacinamide (300 μm of ol/L of final concentration) and Platelet derived growth factor (PDGF-BB) (final concentration 5ng/mL).2 are cultivated under the conditions of 37 DEG C, 5 %CO2 and saturated humidity After hour, whole cells is carefully separately cultured liquid with suction pipe it is transferred in another Tissue Culture Flask and carry out 2 adhere-wall culture 2h, 5mL fresh culture is additionally incorporated in archaeocyte culture bottle simultaneously to continue to cultivate.So can continuous purification up to 3 times or more.It takes more The adherent cell of secondary purifying merges culture, changes within every 3 days liquid 1 time, is disappeared when cell is merged up to 60 % with the pancreatin of 0 .25 % Change secondary culture.

C) stem cell shows that marker is identified

In order to identify the above-mentioned cell being separately cultured, the muscle-derived mescenchymal stem cell in the 3rd generation is taken, culture solution is discarded, It is rinsed one time with PBS.Add 0.25% pancreatin containing EDTA to be digested, terminates digestion with the α-MEM containing 10%FBS.Cell is hanged Liquid is transferred to centrifuge tube, and 1200 r/min are centrifuged 5 min.PBS is transferred to 1mLEP pipe and (about 400 μ is added after washing twice LPBS).The mixing of 40 μ L primary antibodies (CD34, CD45, CD29) is added, is protected from light is incubated for 30 min at room temperature, 1200 r/min centrifugation 5 Min, PBS are rinsed 2 times.The secondary antibody of 100 μ L FITC label is added in every 400 μ L PBS, is protected from light incubation 30 after mixing at room temperature min.1200 r/min of room temperature is centrifuged 5 min, and PBS is washed 2 times, and then plus cell, sample detection expression is resuspended in 400 μ L PBS Amount.

D) stem cell coherency maintains identified for genes

RNA is extracted and reverse transcription:

The muscle-derived mescenchymal stem cell of third generation culture is chosen, 40% density is inoculated with after orifice plate, until cell confluency degree reaches 90% or so, it collects cell and extracts cell RNA progress reverse transcription.RNA is extracted and reverse transcription is using the thin of any company in the market Born of the same parents RNA is extracted and reverse transcription reagent box, and reverse transcription, as primer, is extracted using T18 in strict accordance with operating instruction.

Real-time fluorescence quantitative PCR:

Detection of expression can be completed using disposable type real time fluorescent quantitative instrument, detected gene include target gene (nanog, Sox2 and oct4) and reference gene (actin).

(2) result

A) it is separately cultured system in order to establish pig muscle-derived mescenchymal stem cell, chooses pig longissimus dorsi muscle under aseptic condition, uses glue Protoenzyme digestion, due to use continuous purification with merge cultivate, effectively raise cell purity and density.In addition in culture medium Due to joined fibroblast growth factor 4(FGF4) (final concentration 1ng/mL), niacinamide (300 μm of ol/L of final concentration) and blood platelet Source property growth factor (PDGF-BB), a large amount of energy needed for cell division proliferation and cell Proliferation can be effectively facilitated Amount.

B) in order to carry out epitope identification to the above-mentioned cell being separately cultured, after collecting cell sample, it is separately added into one Cell, the inspection of flow cytometer loading is resuspended after incubation with PBS in the secondary antibody of anti-(CD34, CD45, CD29) and FITC label Survey expression quantity.The results show that CD34 and CD45 are negative, CD29 positive rate is up to 90% or more.

C) correlating markings gene is maintained in order to further carry out stemness to the pig muscle-derived mescenchymal stem cell being separately cultured Detection of expression to the cell extraction RNA cultivated and carries out reverse transcription, with the method for real-time fluorescence quantitative PCR detection nanog, The expression kurtosis of sox2 and oct4 gene.As a result, it has been found that the pig myogenicity mescenchymal stem cell three being separately cultured with the method Stemness maintains gene to have higher gene expression abundance, this shows that cell has stronger multi-lineage potential.

Claims (5)

1. a boar muscle-derived mescenchymal stem cell isolation medium, is cultivated based on the α-MEM culture medium containing fetal calf serum Base, it is characterised in that: in the basal medium also containing fibroblast growth factor 4, niacinamide and platelet derived growth because Son.

2. boar muscle-derived mescenchymal stem cell isolation medium as described in claim 1, it is characterised in that: to contain volume Score is that the α-MEM of 20% fetal calf serum is basic culture medium, also contains final concentration of 0.5-2ng/mL in the basal medium Fibroblast growth factor 4, the niacinamide of final concentration of 100-500 μm of ol/L and the blood platelet source of final concentration of 3-10ng/mL Property growth factor.

3. boar muscle-derived mescenchymal stem cell isolation medium as described in claim 1, it is characterised in that: the basis The volume fraction of fetal calf serum is 20% in culture medium, the concentration of fibroblast growth factor 4 is 1ng/mL, the concentration of niacinamide is The concentration of 300 μm of ol/L and platelet derived growth factor is 5ng/mL.

4. carrying out pig muscle-derived mesenchyma using such as mescenchymal stem cell isolation medium of the claim 1-3 as described in any one The method that stem cell is separately cultured, it is characterised in that: first by collagenase digestion, aseptically from pig muscular tissue Mescenchymal stem cell separation is carried out, then carries out continuous purification using the pig muscle-derived mescenchymal stem cell isolation medium Culture.

5. method as claimed in claim 4, it is characterised in that: operating procedure is as follows:

Step 1: pig longissimus dorsi muscle musculature is cut into small pieces, it is ground into paste homogenate, collected with PBS and washs acquisition is thin Born of the same parents' suspension；Cell suspension is added in the Tissue Culture Flask containing clostridiopetidase A, cultivates 1.5h under the conditions of 37 DEG C, in incubation Repeatedly concussion mixes, and obtains digestive juice；The quality volume fraction of clostridiopetidase A is 2% in the Tissue Culture Flask；

Step 2: mescenchymal stem cell isolation medium is prepared, it is spare；

Step 3: the culture medium that step 2 preparation will be added after digestive juice centrifuge washing obtained by step 1, blows and beats cell with suction pipe, Single cell suspension is made, is then inoculated in the Tissue Culture Flask for being covered with gelatin, 37 DEG C, 5 %CO₂Under the conditions of saturated humidity After cultivating 2h, whole media transfers are continued into adhere-wall culture 2h into another Tissue Culture Flask with suction pipe, while former thin It additionally incorporates fresh culture medium in born of the same parents' culture bottle to continue to cultivate, such continuous purification 3 times or more；Take repeatedly purify it is adherent thin Born of the same parents merge culture, when cell is merged up to 60 % with the pancreatin had digestive transfer culture culture of 0 .25 %.