CN105087462A - Stem cell freezing medium and stem cell freezing method - Google Patents
Stem cell freezing medium and stem cell freezing method Download PDFInfo
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- CN105087462A CN105087462A CN201510443887.6A CN201510443887A CN105087462A CN 105087462 A CN105087462 A CN 105087462A CN 201510443887 A CN201510443887 A CN 201510443887A CN 105087462 A CN105087462 A CN 105087462A
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Abstract
The invention relates to the field of biotechnology, and in particular relates to a stem cell freezing medium and a stem cell freezing method. The stem cell freezing medium consists of DMSO, hydroxyethyl starch and a serum-free medium. The stem cell freezing medium provided by the invention can be used for remarkably reducing injury of the freezing medium on cells, so as to significantly improve the survival rate of the stem cells; and by adopting the serum-free medium, potential pollution caused by serum is avoided.
Description
Technical field
The present invention relates to biological technical field, particularly stem cell cryopreserving liquid and stem cell cryopreserving method.
Background technology
Periodontitis involves the chronic infectious disease of four kinds of Periodontal Supporting Tissue (gum, periodontium, alveolar bone and dental cement), often causes the inflammatory destruction of Periodontal Supporting Tissue.Comparatively common after 35 years old.Common disease is because bacterial plaque, dental calculus, traumatogenic articulation, food impaction, ill fitting prosthesis, mouth breathing etc.Periodontitis has four large features, i.e. periodontal pocket formation, the inflammation of bag wall, frontal resorption, tooth loosen gradually.Serious periodontitis can cause loss of tooth, thus causes masticatory function low and cause maldigestion and gastrointestinal disorder, affects physical and mental health.
Mainly being for the treatment of periodontitis diminishes inflammation, and promotes the regeneration of destroyed periodontal tissue, recovers the normal function of tooth.The periodontitis treatment method of clinical normal employing comprises: periodental non-surgical treatment (scaling, scrape control, root planing), periodontal flap surgery and paradenlal tissue regeneration art.
Periodental non-surgical treatment: scaling, also claim to clean one's teeth, be commonly called as toothwash, it is a kind of method of prevention and therapy odontopathy.Namely the pigment that supragingival calculus, bacterial plaque and facing adhere to is removed with scaling apparatus, and polishing facing, be prevent bacterial plaque and dental calculus from depositing again, prevent and treat the measure of periodontopathy.Also a part of subgingival calculus be connected with supragingival calculus in gingival sulcus also should be disposed in the lump when scaling.Different according to apparatus used, supragingival scaling is divided into hand instrument scaling method and ultrasonic cavitron scaling method.For gingivitis patients, within every 6 ~ 12 months, do a scaling, effectively can safeguard periodontal health.Subgingival curettage, i.e. root planing, it is that subgingival debridement apparatus by hand stretches in periodontal pocket to remove and to be attached in periodontal pocket on root face and the subgingival calculus embedded in dental cement and bacterial plaque, and strike off the pathology dental cement that root surface is subject to endotoxin contamination, thus form smooth, clean root face, make root mask have biocompatible surface, be conducive to attachment and the new life of periodontal tissue.Root planing shall not be applied to healthy periodontal loci, in order to avoid cause periodontal attachment loss.
Periodontal flap surgery: refer to adopt different operative incision, by the separate tissue of gum with below, forms gingiva tissue lobe, and the root face of exposure diseased region and alveolar bone, provide debridement approach and visuality.After striking off pathological tissues and bacterial plaque dental calculus, gingival flap is resetted and goes up in place and sew up, reach the object eliminated periodontal pocket or periodontal pocket is shoaled.
Paradenlal tissue regeneration art: the alveolar bone district implantable artificial bone lacked, and cover special biological guiding film, grow on root face with making the cell selective on periodontium, thus reach periodontium, alveolar bone and cemental regeneration.Planted and biomembranous technology by this bone, recover the alveolar bone, gum and other the periodontal tissue that have destroyed, complete again stablizing of tooth.
Above method cannot repair periodontal attachment and the alveolus tissue of damage, can not meet at present clinically to the requirement that Oral and maxillofacial defect reparative regeneration and function, profile are recovered, secondly periodontitis treatment usually controls bacterial plaque growth by antibiosis, carries out the pharmacological agent of whole body and local.But antibiotic side effect is more, pathogenic micro-organism also can produce resistance.
Dental pulp stem cell (dentalpulpstemcells, DPSCs) is that the class be present in tooth pulp tissue has self, multiplication capacity is strong, and the mescenchymal stem cell of Multidirectional Differentiation ability.Dental pulp stem cell has the potential of Multidirectional Differentiation, and it through the induction of different cytokines, can also be divided into the clone types such as fat, bone, cartilage, muscle, blood vessel endothelium, liver, nerve except forming the extracellular of Mineral nodules ability.It is reported, dental pulp stem cell can play good repair in periodontitis treatment, and mechanism of action is: by implanting local and being divided into defect cell, secrete cytokines, and chemotactic stem cell, to local, suppresses local inflammation, promotes that local vascular is newborn.
At present, formula for the cells frozen storing liquid of dental pulp stem cell is 10%DMSO+90%FBS, this cells frozen storing liquid can produce certain damaging action to it in the process of preserving or transporting dental pulp stem cell, makes dental pulp stem cell motility rate not reach desirable level, affects its result for the treatment of.
Summary of the invention
In view of this, the invention provides stem cell cryopreserving liquid and stem cell cryopreserving method.This stem cell cryopreserving liquid significantly can reduce the damaging action of frozen storing liquid to cell, thus can significantly improve the motility rate of stem cell; The present invention selects serum free medium, avoids the potentially contaminated that serum brings.。
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of stem cell cryopreserving liquid, comprise DMSO, hydroxyethylamyle and serum free medium.
As preferably, in volumn concentration, DMSO accounts for 5% ~ 15%; In mass percentage, hydroxyethylamyle accounts for 1% ~ 3%; Surplus is serum free medium.
Preferably, in volumn concentration, DMSO accounts for 10%; In mass percentage, hydroxyethylamyle accounts for 1%; Surplus is serum free medium.
Preferably, in volumn concentration, DMSO accounts for 15%; In mass percentage, hydroxyethylamyle accounts for 2%; Surplus is serum free medium.
Preferably, in volumn concentration, DMSO accounts for 5%; In mass percentage, hydroxyethylamyle accounts for 3%; Surplus is serum free medium.
In embodiments more provided by the invention, stem cell cryopreserving liquid is used for frozen dental pulp stem cell.But the present invention is not limited to frozen dental pulp stem cell, the stem cell of other kind also can be frozen with stem cell cryopreserving liquid provided by the invention.
Present invention also offers a kind of stem cell cryopreserving method, adopt the frozen stem cell of stem cell cryopreserving liquid of the present invention; This stem cell cryopreserving liquid comprises DMSO, hydroxyethylamyle and serum free medium; As preferably, in volumn concentration, DMSO accounts for 5% ~ 15%; In mass percentage, hydroxyethylamyle accounts for 1% ~ 3%; Surplus is serum free medium; Preferably, in volumn concentration, DMSO accounts for 10%; In mass percentage, hydroxyethylamyle accounts for 1%; Surplus is serum free medium; Preferably, in volumn concentration, DMSO accounts for 15%; In mass percentage, hydroxyethylamyle accounts for 2%; Surplus is serum free medium; Preferably, in volumn concentration, DMSO accounts for 5%; In mass percentage, hydroxyethylamyle accounts for 3%; Surplus is serum free medium; In embodiments more provided by the invention, stem cell cryopreserving liquid is used for frozen dental pulp stem cell.
As preferably, the density of stem cell is (0.1 ~ 10) × 10
6cell/mL.
Preferably, the density of stem cell is 1 × 10
6cell/mL.
As preferably, frozen temperature≤-80 DEG C.
In embodiments more provided by the invention, frozen temperature is-180 DEG C.
In embodiments more provided by the invention, stem cell is dental pulp stem cell.
As preferably, stem cell is that P3 is for stem cell.
The invention provides stem cell cryopreserving liquid and stem cell cryopreserving method.This stem cell cryopreserving liquid comprises DMSO, hydroxyethylamyle and serum free medium.The present invention at least has one of following advantage:
Stem cell cryopreserving liquid provided by the invention significantly can reduce the damaging action of frozen storing liquid to cell, thus can significantly improve the motility rate of stem cell;
The present invention selects serum free medium, avoids the potentially contaminated that serum brings.
Accompanying drawing explanation
Cell viability histogram after Fig. 1 gives instructions in reply and revives;
Fig. 2 shows flow cytomery blank group cell-surface antigens expression;
Fig. 3 shows flow cytomery experimental group cell-surface antigens expression.
Embodiment
The invention discloses stem cell cryopreserving liquid and stem cell cryopreserving method, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
In stem cell cryopreserving liquid provided by the invention and stem cell cryopreserving method, agents useful for same, instrument all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 ~ 3 dental pulp stem cell
(1) separation and Culture dental pulp stem cell:
After family members agree to, collects the deciduous incisor tooth that 6 years old-10 years old children pull out because of delay clinically, require do not have tooth prosthesis to become and necrosis.Being put in 4 DEG C after pulling out immediately meets in the cold PBS centrifuge tube be equipped with containing dual anti-(100 μ g/mL penicillin, 100 μ g/mL Streptomycin sulphates), takes out and use in 24 hours.
Take out tooth, utilize and repeatedly rinse three times containing dual anti-PBS solution, tooth is wrapped in clamp schizodont tooth in sterile gauze, Exposed Pulp tissue; With aseptic nipper gripping pulp tissue, the pulp tissue of excision root tip 1mm.According to pulp tissue's size, with ophthalmology curved scissors, pulp tissue is cut into 1mm
3, be placed in 50mL centrifuge tube.Add the 5g/LI Collagenase Type of 10 times of volumes, fully mix after sealing, be transferred in Tempeerature-constant air shaking table, 37 DEG C, 200R digests 20min, then use the centrifugal 3min of 2000rpm.Abandon supernatant., use 30mLPBS washing and precipitating, repeatedly blow and beat discrete cellular agglomerate, by the cell screen filtration of 70um, to obtain single discrete cell, the centrifugal 5min of 1000rpm.Abandon supernatant, precipitation serum free medium is resuspended, with (0.5 ~ 1.5) × 10
4/ cm
2be seeded in six orifice plates, every hole adds 2mL serum free medium, add Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations are 10ng/mL) again, mixing cell suspension, is put in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%.Every 3d changes liquid 1 time, and about 7-14d can observe the formation of clone under the microscope, and when Growth of Cells converges to 80%-90%, 0.125% trypsin digestion and cell, carries out Secondary Culture.
(2) amplification cultivation of dental pulp stem cell and frozen:
After the cell confluency degree of above-mentioned primary cell reaches 80%-90%, use 0.1%-0.25% tryptic digestion, with the centrifugal 5min of 1000-1500rpm, according to (0.5-1.5) × 10
4cell/cm
2cell density goes down to posterity, and the substratum of Secondary Culture is serum free medium+Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations is for being 10ng/mL).Passage is to the 3rd generation, and cell carries out frozen.Cell cryopreservation formula is:
Table 1 dental pulp stem cell frozen storing liquid is filled a prescription
Cell is frozen in 2mL cryopreservation tube, and cell cryopreservation density is 1 × 10
6cell/mL, often pipe 1.5mL frozen storing liquid.Detect the stem-cell marker expression such as frozen front cell quantity, cell viability, flow cytometer detection CD73, CD90, CD45, HLA-DR, simultaneously microorganism detection freeze-stored cell suspension (detecting fungi, cell, mycoplasma).The substratum of Secondary Culture is serum free medium+Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations is for being 10ng/mL).
(3) dental pulp stem cell cell recovery amplification cultivation:
Frozen P3 cell is recovered, according to (0.5-1.5) × 10
4cell/cm
2inoculum density is inoculated in 15cm culture dish, amounts to inoculation 10 15cm culture dish.Substratum is serum free medium+Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations is for being 10ng/mL).Be put in 37 DEG C, humidity is cultivate 2-3 days in the CO2gas incubator of 95%.
(4) dental pulp stem cell is collected:
When cell confluency degree reaches 80%-90%, draw substratum, culture dish is cleaned 2 times with physiological saline, use 0.125% trypsin digestion and cell again, when attached cell is shrunk to circle, can trypsinase be drawn, add physiological saline piping and druming cell, be collected in 50mL centrifuge tube, with the centrifugal 5min of 1000-1500rpm, remove supernatant, collecting precipitation, after again cleaning cell 2 times with physiological saline, cross 70 μm of cell sieves, carry out cell counting subsequently, centrifugal rear physiological saline is resuspended, divides and is filled in 1mL syringe.Detect Cell viability.
(5) test-results:
After recovery, Cell viability is in table 2, Fig. 1:
Table 2 recover after Cell viability
From above-mentioned test-results, the stem cell cryopreserving liquid that the embodiment of the present invention 1 ~ 3 provides significantly can reduce the damaging action of frozen storing liquid to cell, thus can significantly improve the motility rate of dental pulp stem cell.
The preparation of embodiment 4 dental pulp stem cell injection liquid
(1) separation and Culture dental pulp stem cell
Collect the healthy tooth coming off with servant and extract for 30 years old, PBS cleans dental surface dirt, with clamp schizodont tooth, and Exposed Pulp tissue; With aseptic nipper gripping pulp tissue.Shred, place in 50mL centrifuge tube, add the 3g/LI Collagenase Type of 10 times of volumes, fully mix after sealing, be transferred in Tempeerature-constant air shaking table, 37 DEG C, 200R digests about 10-20min, add isopyknic perfect medium and stop digestion, repeatedly blow and beat discrete cellular agglomerate, by the cell screen filtration of 70 μm, to obtain single discrete cell, the centrifugal 5min of 1000rpm.Abandon supernatant, by 30mLPBS washing and precipitating 1 time, the centrifugal 5min of 1000rpm.Precipitation serum free medium is resuspended, with 0.5 ~ 1 × 10
4/ cm
2be seeded in six orifice plates, every hole adds 2mL serum free medium, add Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations are 10ng/mL) again, mixing cell suspension, is put in 37 DEG C, humidity is cultivate in the CO2gas incubator of 95%.Every 3d changes liquid 1 time, and about 7-14d can observe the formation of clone under the microscope, and when Growth of Cells converges to 80%-90%, 0.125% trypsin digestion and cell, carries out Secondary Culture.
(2) amplification cultivation of dental pulp stem cell and frozen
After above-mentioned primary cell cell confluency degree reaches 80%-90%, use 0.1%-0.25% tryptic digestion, with 1000 centrifugal 5min, according to (0.5 ~ 1) × 10
4cell/cm
2cell density goes down to posterity, and the substratum of Secondary Culture is serum free medium+Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations is for being 10ng/mL).When passage is to the 3rd generation, cell confluency degree reach 80% ~ 90% can carry out frozen.Cell 0.125% tryptic digestion centrifugal collecting cell, draws 20 μ L suspensions and carries out cell quantity, cell viability detection.Carry out cell cryopreservation subsequently.Cell cryopreservation formula is: 10%DMSO, 1% hydroxyethylamyle, serum free medium.Cell is frozen in 2mL cryopreservation tube, and cell cryopreservation density is 1 × 10
6cell/mL, often pipe 1.5mL frozen storing liquid.Draw the frozen suspension of 1mL and carry out microorganism detection (detecting fungi, cell, mycoplasma).
Get respectively the 3rd generation cell, with 0.125% tryptic digestion collecting cell, after counting, often pipe adds 2 × 10
5cell count, dye solution washes 1 time, the centrifugal 5min of 1000rpm; Abandon supernatant, with dye solution piping and druming mixing cell; Add each 2 μ L of CD45, CD73, CD90 and HLA-DRA antibody, and set a pipe as blank; At 4 DEG C, lucifuge reaction 15-20min; Dye solution is washed once, the centrifugal 5min of 1000rpm; The cell of direct mark abandons supernatant, and lucifuge adds the sample-loading buffer of 500 μ L, and mixing, with 200 eye mesh screen filtration cell samples, flow cytomery cell-surface antigens expression, the results are shown in Figure 2,3.
All present feminine gender from Fig. 2,3, CD45 (white corpuscle is positive), HLA-DR (MHC-II quasi-molecule) for expressing at dental pulp stem cell, dental pulp stem cell surface marker CD73, CD90 all presents the positive simultaneously.This result meets dental pulp stem cell surface markers expression characteristic.
(3) preparation of dental pulp stem cell recovery cultivation and injection liquid
Frozen P3 cell is recovered, and detects cell quantity and vigor, according to 0.5 × 10
4cell/cm
2inoculum density is inoculated in 15cm culture dish, amounts to inoculation 10 15cm culture dish.Substratum is serum free medium+Urogastron EGF and Basic Fibroblast Growth Factor bFGF (both final concentrations is for being 10ng/mL).Be put in 37 DEG C, humidity is cultivate 2-3 days in the CO2gas incubator of 95%.When cell confluency degree reaches 80%-90%, draw substratum, culture dish is cleaned 2 times with physiological saline, use 0.125% trypsin digestion and cell again, when attached cell is shrunk to circle, can trypsinase be drawn, add physiological saline piping and druming cell, be collected in 50mL centrifuge tube, with the centrifugal 5min of 1000-1500rpm, remove supernatant, collecting precipitation, after again cleaning cell 2 times with physiological saline, cross 70 μm of cell sieves, carry out cell counting and cell viability subsequently, centrifugal rear physiological saline is resuspended, divides and is filled in 1mL syringe.
(4) acute toxicity test of dental pulp stem cell injection liquid
By dental pulp stem cell injection liquid to healthy mice according to 1 × 10
6cell, 5 × 10
6cell, 10 × 10
6cell/KG continuous use 2 weeks (every 3 days once), after discontinuing medication and drug withdrawal carry out physical signs detection respectively after 2 weeks, result shows: the index such as hair, body weight, hepatic and renal function, blood sugar, blood fat of dental pulp stem cell injection liquid to mouse is all without exception, mice organs official is without considerable change, illustrate that dental pulp stem cell injection liquid is little to small white mouse long-term prescription toxicity, use safety.
(5) experimentation on animals of dental pulp stem cell injection liquid
30 of Periodontitis Model rats are divided into 3 groups at random: physiological saline blank group, positive controls (Minocycline ointment, outer painting), dental pulp stem cell group (being female 5, male 5).Administration adopts gum district local injection, and every day 2 times, 10d was 1 course for the treatment of.Select the index that gingival index (GI), SBI (SBI), periodontal index (PI), plaque index (PLI) 4 indexes are observed as curative effect after Experimental Periodontitis rat drug treatment.
Result is as shown in the table:
Curative effect observation index detected result after table 3 rat drug treatment
| Physical signs | GI | SBI | PI | PLI |
| Blank group | 3.1+0.43 | 60.2±0.56 | 7.23±1.43 | 2.98±0.45 |
| Positive controls | 1.92±0.33 | 3.45±0.63 | 5.9±1.25 | 2.28±0.49 |
| Sample control group | 1.26±1.18 | 2.45±0.42 | 3.43±1.17 | 2.11±0.67 |
From the above results, the result for the treatment of of dental pulp stem cell injection liquid provided by the invention is better.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a stem cell cryopreserving liquid, is characterized in that, comprises DMSO, hydroxyethylamyle and serum free medium.
2. stem cell cryopreserving liquid according to claim 1, is characterized in that, in volumn concentration, described DMSO accounts for 5% ~ 15%; In mass percentage, described hydroxyethylamyle accounts for 1% ~ 3%; Surplus is serum free medium.
3. stem cell cryopreserving liquid according to claim 2, is characterized in that, in volumn concentration, described DMSO accounts for 10%; In mass percentage, described hydroxyethylamyle accounts for 1%; Surplus is serum free medium.
4. stem cell cryopreserving liquid according to claim 1, is characterized in that, described stem cell cryopreserving liquid is used for frozen dental pulp stem cell.
5. a stem cell cryopreserving method, is characterized in that, adopts the frozen stem cell of stem cell cryopreserving liquid according to any one of Claims 1-4.
6. stem cell cryopreserving method according to claim 5, is characterized in that, the density of described stem cell is (0.1 ~ 10) × 10
6cell/mL.
7. stem cell cryopreserving method according to claim 6, is characterized in that, the density of described stem cell is 1 × 10
6cell/mL.
8. stem cell cryopreserving method according to claim 5, is characterized in that, described frozen temperature≤-80 DEG C.
9. stem cell cryopreserving method according to claim 5, is characterized in that, described stem cell is dental pulp stem cell.
10. the stem cell cryopreserving method according to any one of claim 5 to 9, is characterized in that, described stem cell is that P3 is for stem cell.
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| CN105754934A (en) * | 2016-03-08 | 2016-07-13 | 大连大学 | Dental pulp stem cell, preparation method thereof and related bone tissue engineering material |
| CN106359368A (en) * | 2016-09-30 | 2017-02-01 | 广州赛莱拉干细胞科技股份有限公司 | Cell cryoprotectant and cryopreservation method |
| CN108029678A (en) * | 2017-12-28 | 2018-05-15 | 重庆斯德姆生物技术有限公司 | Cell preservation method and cell transportation resources |
| CN108056095A (en) * | 2017-12-28 | 2018-05-22 | 重庆斯德姆生物技术有限公司 | A kind of fat mesenchymal stem cell transport protection liquid and its application |
| CN108450459A (en) * | 2016-03-06 | 2018-08-28 | 李倩 | A kind of cells frozen storing liquid of human adipose-derived stem cell |
| CN114532330A (en) * | 2022-02-22 | 2022-05-27 | 张俊杰 | Cryopreservation method of dental pulp mesenchymal stem cells |
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| CN108450459A (en) * | 2016-03-06 | 2018-08-28 | 李倩 | A kind of cells frozen storing liquid of human adipose-derived stem cell |
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| CN108029678A (en) * | 2017-12-28 | 2018-05-15 | 重庆斯德姆生物技术有限公司 | Cell preservation method and cell transportation resources |
| CN108056095A (en) * | 2017-12-28 | 2018-05-22 | 重庆斯德姆生物技术有限公司 | A kind of fat mesenchymal stem cell transport protection liquid and its application |
| CN114532330A (en) * | 2022-02-22 | 2022-05-27 | 张俊杰 | Cryopreservation method of dental pulp mesenchymal stem cells |
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