CN101914496A - Simplified method for preparing umbilical mesenchymal stem cells in large scale - Google Patents

Abstract

The invention relates to a method for preparing umbilical mesenchymal stem cells in a large scale. In the method, although umbilical cord cutouts are larger, the primary culture time is greatly shortened. A medium contains bFGF and FBS, promotes adherence growth of mesenchymal stem cells and does not influence differentiation capacity of the stem cells. After 8 to 9 days, about 80 percent of cells are fused, the cells grow fast and the generation time is short.

Description

A kind of large-scale preparation method of umbilical cord mesenchymal stem cells of simplification

Technical field

The invention belongs to biological technical field, be specifically related to a kind of large-scale preparation method of mescenchymal stem cell.

Background technology

(mesenchymal stem cells is the important member of stem cell family MSC) to mescenchymal stem cell, derives to grow early stage mesoderm and ectoderm.MSC finds in marrow at first, because of it has multidirectional differentiation potential, hematopoiesis support and promotes characteristics such as stem cell implantation, immunoregulation and self-replacation to be subjected to people's attention day by day.As mescenchymal stem cell in vivo or under the external specific inductive condition, can be divided into multiple histocytes such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium, still have multidirectional differentiation potential after continuous passage cultivation and the freezing preservation, can be used as the ideal seed cell and be used for injuries of tissues and organs reparation old and feeble and that pathology causes.

The mescenchymal stem cell clinical application is in solving multiple disease in the blood system, diabetes and diabetic foot, cardiovascular disorder, liver cirrhosis, Spinal injury, nervous system disorderss such as Parkinson's disease, meniscus of knee joint partly excises injury repairing, aspects such as autoimmune disorder have obtained important breakthrough, have saved the life of many sufferers.

Mescenchymal stem cell is distributed widely in the marrow, periosteum, spongy bone, fat, synovial membrane, skeletal muscle, tire liver, deciduous teeth, umbilical cord, Cord blood of fetus and adult, wherein the mescenchymal stem cell quality height in umbilical cord source, pure, quantity is many.The mescenchymal stem cell in umbilical cord source not only can become the desirable surrogate of mesenchymal stem cells MSCs, and has bigger application potential.

Umbilical cord mesenchymal stem cells is expressed the peculiar molecular marker of multiple embryonic stem cell, have that differentiation potential is big, multiplication capacity is strong, immunogenicity is low, draw materials conveniently, the features such as restriction of amoral ethics problem, therefore might become the multipotential stem cell of tool potential applicability in clinical practice.But the separation and Extraction process of umbilical cord mesenchymal stem cells is loaded down with trivial details and extraction efficiency is low, and the scale preparation of mescenchymal stem cell has become the bottleneck of its widespread use.

Mescenchymal stem cell preparation at present to be adopted enzyme digestion and organizes adherent method to prepare.It is after umbilical cord tissue is cut into fragment that enzyme digestion extracts umbilical cord mesenchymal stem cells, with trypsinase and collagenase digesting 1-2 hour, stop enzymic digestion with the substratum that contains foetal calf serum, washed cell discards digestive ferment then, and the cell inoculation after the washing is cultivated amplification to cultivating in the vessel.Washed cell takes about 1 hour, and pair cell damage is heavier in the washing process, and complex operation step is easily polluted, and the damage of digestive ferment pair cell is also heavier, causes finally that the cell viability that obtains is low, propagation is slow.Organizing adherent method is more a kind of method of using at present, and traditional adherent method of organizing is that umbilical cord tissue is shredded, and requires to organize the degree that shreds very high, cut to the meat gruel shape, shear time more than 30min, time and effort consuming; Inoculation culture behind the centrifugal flush away tissue juice, the mescenchymal stem cell adherent growth is slower, needs 10-14 days more.

Enzyme digestion prepares mescenchymal stem cell length consuming time, in the operating process pair cell damage bigger, make and separate that the mescenchymal stem cell vigor that obtains is low, propagation is slow.It is very high to organize adherent method requirement umbilical cord tissue to shred degree, the operation effort, and cell attachment is slow, and growth is slow.

Summary of the invention

The inventor is at above-mentioned shortcoming, extensively consult a large amount of domestic and foreign literatures, on the basis of existing mescenchymal stem cell technology of preparing, the adherent method technology of preparing is organized in improvement, the tissue block shear history is no longer required great effort, and in nutrient solution, add bFGF (Prostatropin), can accelerate the adherent of cell and growth, just make the whole former time of supporting of being commissioned to train greatly shorten by original 10-14 days.

The present invention adopts following technical scheme:

The invention provides a kind of preparation method of umbilical cord mesenchymal stem cells, may further comprise the steps:

1) fresh umbilical cord is with the flushing of PBS damping fluid; Described PBS damping fluid contains 100kU/L penicillin and 100mg/L Streptomycin sulphate;

2) the long umbilical cord of clip 6-8cm is cut into the long segment of 2cm with umbilical cord, washes repeatedly with the PBS damping fluid; Described PBS damping fluid contains 100kU/L penicillin and 100mg/L Streptomycin sulphate;

3) the umbilical cord tissue piece is shredded into 2-3mm ³Fritter;

4) will shred tissue and add the washing of L-DMEM substratum, under the 650g condition centrifugal 5 minutes, abandon supernatant;

5) with tissue block and substratum 2.5-3 by volume: 1 ratio adds substratum, and the mixing tissue block is seeded in the Tissue Culture Dish, puts incubator and cultivates; Described substratum is for containing the L-DMEM substratum of 1-10ng/ml Prostatropin (bFGF) and volume percent 10%-15% foetal calf serum (FBS);

6) add the described substratum of step 5 after 24 hours.

7) changed a subculture in per 3 days, change the L-DMEM substratum that contains volume ratio 10% foetal calf serum after the 6th day, went down to posterity when cell reached about 80% fusions to 8-9 days; The substratum that goes down to posterity is the L-DMEM substratum that contains volume ratio 10% foetal calf serum;

8) went down to posterity once in per 3 days after.

Description of drawings

Fig. 1: the cellular form of the mescenchymal stem cell in people's umbilical cord source: (A-C) be the cellular form in the 4th, 5,7 days former generations of mescenchymal stem cell of adopting that method of the present invention obtains; (D-F) be the cellular form of the mescenchymal stem cell first-generation after the 1st, 2,3 days; (G) be the 3rd day cellular form of the mescenchymal stem cell s-generation; (H-J) be the 1st, 2,3 days cellular form of the mescenchymal stem cell third generation.

Fig. 2: the cell growth curve of human umbilical cord mesenchymal stem cells

Fig. 3: the flow cytometry cell phenotype is analyzed

Technique effect

1, the umbilical cord flushing thoroughly contains antibiotic in the flushing liquor, can maximum wash crude removal and remaining blood, avoids polluting.

2, umbilical cord shearing piece is bigger, and it is time saving and energy saving to shear. The former culture time of shortening that in the case still can be a large amount of.

3, determine the mixed best ratio of adding culture medium when organizing piece of sparing, avoided the not enough or unnecessary impact that cell is cultivated of culture medium.

4, culture medium is for containing the fresh complete culture medium L-DMEM of bFGF (concentration 1-10ng/ml) and 10%-15%FBS. Mesenchymal stem cells adherent growth between the low concentration bFGF that adds had both promoted does not affect again the differentiation capability of stem cell.

5, change culture medium after the 6th day and be (L-DMEM+FBS), went down to posterity when cell reached about 80% fusions to 8-9 days, the cell growth is fast, and the generation time is short.

The tool present embodiment

1. fresh umbilical cord, after the detection of process virus was qualified, ((pH7.2～7.4) contained NaCl 137mmol/L, KCl 2.7mmol/L, Na with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate ₂HPO ₄4.3mmol/L, KH ₂PO ₄1.4mmol/) flush away dirt and blood.

2. the about 6-8cm umbilical cord of clip is cut into segment about 2cm with umbilical cord, washes away umbilical vein and endarterial remaining blood with the PBS damping fluid that contains 100kU/L penicillin and 100mg/L Streptomycin sulphate as far as possible.

3. with umbilical cord scissors the umbilical cord tissue piece is shredded into 2-3mm ³Fritter.

4. will shred tissue adds the L-DMEM substratum (Gibco company, USA) washing under the 650g condition centrifugal 5 minutes, is abandoned supernatant.

5. according to 2.5-3: 1 ratio (tissue block: substratum, volume ratio) adds substratum (L-DMEM+FBS+bFGF) (promptly, the L-DMEM substratum that contains 1-10ng/ml bFGF and volume percent 10%-15%FBS) the mixing tissue block is seeded in the Tissue Culture Dish, puts 37 ℃, 5%CO ₂The saturated humidity incubator cultivate.Described FBS is a foetal calf serum, and bFGF is a Prostatropin.

6.24 add the described substratum of step 5 after hour.

7. changed a subculture in per 3 days, see that spindle cell climbs out of around the 5-6 days tissue block.Change substratum after the 6th day and be (L-DMEM+ volume ratio 10%FBS), went down to posterity when cell reached about 80% fusions to 8-9 days; The substratum that goes down to posterity is L-DMEM+ volume ratio 10%FBS.

8. went down to posterity once in per 3 days later on, identify to the third generation.

Fig. 1, Fig. 2, Fig. 3 are seen in the cellular form of the mescenchymal stem cell in each stage, upgrowth situation, cell phenotype analysis.Control experiment (conventional organization piece adherent method)

1. get umbilical cord, stroke-physiological saline solution flushing crude removal and blood;

2. aseptic hammer-shears umbilical cord is to 1-2mm ³Size.

3. tissue block is transferred in the centrifuge tube of 50ml, adds the PBS damping fluid 40ml contain 1% mycillin, room temperature under the 250g condition centrifugal 5 minutes is abandoned supernatant.

The tissue block that obtains with containing the DMEM culture medium culturing of volume ratio 10%, changed once in per 3 days and to cultivate.

5.7 have fusiformis and polygon cell to climb out of around it left and right sides tissue block, 10-14 days left and right sides cells just reach about 80% and merge, and go down to posterity with 0.25% trysinization.

Experimental result: method of the present invention, contain microbiotic in the umbilical cord washing fluid, can maximum flush away dirt and remaining blood, avoided pollution; Though the umbilical cord scissors stripping and slicing is bigger, be commissioned to train the time of supporting but still shortening that can be a large amount of is former; Add the optimum proportion of substratum when having determined the mixing tissue block, substratum is for containing the fresh perfect medium L-DMEM of bFGF (concentration 1-10ng/ml) and 10%-15%FBS, both promoted the mescenchymal stem cell adherent growth, do not influence the differentiation capability of stem cell again, promptly reaching about 80% to 8-9 days cells merges, the cell growth is fast, and the generation time is short.

Claims (2)

1. the preparation method of a umbilical cord mesenchymal stem cells is characterized in that may further comprise the steps:

1) fresh umbilical cord is with the flushing of PBS damping fluid;

2) the long umbilical cord of clip 6-8cm is cut into the long segment of 2cm with umbilical cord, washes repeatedly with the PBS damping fluid;

3) the umbilical cord tissue piece is shredded into 2-3mm ³Fritter;

4) will shred tissue and add the washing of L-DMEM substratum, under the 650g condition centrifugal 5 minutes, abandon supernatant;

5) with tissue block and substratum 2.5-3 by volume: 1 ratio adds substratum, and the mixing tissue block is seeded in the Tissue Culture Dish, puts incubator and cultivates; Described substratum is the L-DMEM substratum that contains 1-10ng/ml Prostatropin and volume percent 10%-15% foetal calf serum;

6) add the described substratum of step 5 after 24 hours;

7) changed a subculture in per 3 days, change the L-DMEM substratum that contains volume ratio 10% foetal calf serum after the 6th day, went down to posterity when cell reached about 80% fusions to 8-9 days; The substratum that goes down to posterity is the L-DMEM substratum that contains volume ratio 10% foetal calf serum;

8) went down to posterity once in per 3 days after.

2. the method for claim 1 is characterized in that described PBS damping fluid contains 100kU/L penicillin and 100mg/L Streptomycin sulphate.

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