CN105211053A - Cryopreservation solution for tendon stem cells derived from human achilles tendon and preparation method thereof - Google Patents
Cryopreservation solution for tendon stem cells derived from human achilles tendon and preparation method thereof Download PDFInfo
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- CN105211053A CN105211053A CN201510727859.7A CN201510727859A CN105211053A CN 105211053 A CN105211053 A CN 105211053A CN 201510727859 A CN201510727859 A CN 201510727859A CN 105211053 A CN105211053 A CN 105211053A
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Abstract
The invention relates to the technical field of stem cells, in particular to a cryopreservation solution for tendon stem cells derived from human achilles tendon and a preparation method thereof. The cryopreservation solution of the tendon stem cells derived from the human achilles tendon consists of conditioned medium and dimethyl sulfoxide. The preparation method of the frozen stock solution of the tendon stem cells derived from the human achilles tendon comprises the following steps: (1) culturing cells; (2) preparing a conditioned medium; (3) according to the following steps of 9: 1 volume ratio the conditioned medium was mixed with dimethyl sulfoxide. The invention can reduce the cost of cell cryopreservation to a certain extent, can avoid the introduction of heterologous substances, and has higher clinical safety compared with the conventional cell cryopreservation solution. The cryopreservation solution for the tendon stem cells derived from the human achilles tendon has small damage effect on the cells in the process of cell cryopreservation, has high cell survival rate, has a cryopreservation effect obviously superior to that of the conventional cell cryopreservation solution, and can be used for long-term preservation and application of the tendon stem cells derived from the human achilles tendon.
Description
Technical field
The present invention relates to stem cells technology field, be specifically related to cryopreserving liquid of a kind of people's heel string source tendon stem cell and preparation method thereof.
Background technology
Stem cell is the multipotential cell that a class has self-renewal capacity (self-renewing).Under certain condition, it can be divided into several functions cell, and medical circle is called " general-purpose cell ".Developmental stage residing for stem cell is divided into embryonic stem cell (embryonicstemcell, ES cell) and adult stem cell (somaticstemcell).Potentiality of development according to stem cell is divided three classes: myeloid-lymphoid stem cell (totipotentstemcell, TSC), multipotential stem cell (pluripotentstemcell) and unipotent stem cell (unipotentstemcell) (specially energy stem cell).
Caput femoris necrosis is orthopaedics common disease, frequently-occurring disease, and the course of disease is long and disability rate is high.How effectively to treat femoral head downright bad, prevent further developing of disease, protection femoral head, delays the operating time of hip replacement, is the emphasis for the treatment of.Although the method for hip-preserving surgery is a lot of at present, as the fibular autograft, bone marrow mescenchymal stem cell, surface replacement etc. of core decompression, vascular pedicle, all obtains certain curative effect, do not have a kind of technology to be satisfied with completely.Since 2007, Chinese scholars is separated and has turned out tendon stem cell (TDSCs) from the tendon of humans and animals, and this cell has multi-lineage potential (primary differentiation is Gegenbaur's cell, chondroblast, adipocyte) and self-renewal capacity.Tendon stem cell has clear and definite stem cell properties, and these characteristics are applicable to the pathologic process of repairing caput femoris necrosis, and being therefore expected to becomes the new seed cell for the treatment of caput femoris necrosis, therefore studies people's heel string source tendon stem cell important in inhibiting.
Stem cell has potential medical value and using value, and cell preservation technique is the basis realizing these potential values.After stem cell storage refers to and isolated and cultured from different tissues by stem cell, then through Testing and appraisal, then will preserve, so that stem cell can be used for when clinical needs feeding back to patient, reach the object of disease therapy.Cell preservation method conventional at present has cultivation and frozen two kinds of modes.Wherein cultivate preservation, take time and effort, program is loaded down with trivial details, and in the process of cultivating, particularly during long term subculture, cell easily morphs, and cell characteristics is lost, and loses the value of preservation.And another kind of cell preservation technique and cell cryopreservation, utilize Cryopreservation Technology cell to be placed in-196 DEG C of liquid nitrogen Cord blood, cell can be made temporarily to depart from growth conditions and be saved by its cell characteristics, when needs, recovery cell is used for experiment more like this.And moderately preserve a certain amount of cell, can prevent from making cell lose kind because of just contaminated in cultured cells or other accidents, serve the effect of cell conservation.In addition, the form of cell cryopreservation can also be utilized buy, post and give, exchange and transport some cell.In medium, protectant is added during cell cryopreservation--the glycerine of final concentration 5%-15% or dimethyl sulfoxide (DMSO) (DMSO); freezing point of solution can be made to reduce, and in addition under slow freezing condition, ICW appears; decrease ice crystal to be formed, thus avoid cellular damage.Adopt the method for " freeze slowly and melt soon " cell survival can be ensured preferably.Standard chilling rate starts as-1 to-2 DEG C/min, when temperature can be accelerated lower than when-25 DEG C, and afterwards can direct plunge into Liquid Nitrogen interior (-196 DEG C) to-80 DEG C.
Normally be made up of the material such as hyclone and dimethyl sulfoxide (DMSO) at present conventional cells frozen storing liquid or commercially available cells frozen storing liquid, but hyclone belongs to heterologous material, complicated component, and there is the risk introducing pollution and anaphylactogen, be not suitable for clinical practice.Particularly in cell therapy, the existence of heterologous protein, may cause unknown bad reaction, have a strong impact on treatment results.
Conditioned medium (conditionmedium, CM): refer in cell cultivation process, this cell may secretion activity material in culture fluid, wherein cell factor is one of main active substance, this cultivated certain cell or tissue after, the culture fluid containing cell secreta is just called conditioned medium.Utilize conditioned medium effectively can improve the success rate of cell or tissue culture in vitro, and the conditioned medium of separate sources have different promotions or inhibitory action to different cell, tissue.
Summary of the invention
The present invention is directed to problems of the prior art, provide a kind of clinical safety high, well can keep again the cells frozen storing liquid of frozen activity simultaneously, for cryopreserved human heel string source tendon stem cell.
The present invention also provides a kind of method preparing the cryopreserving liquid of described people's heel string source tendon stem cell.
The present invention is achieved through the following technical solutions this object:
A cryopreserving liquid for people's heel string source tendon stem cell, is made up of conditioned medium and dimethyl sulfoxide (DMSO).
As preferably, the cryopreserving liquid conditional culture fluid of described people's heel string source tendon stem cell and the volume ratio of dimethyl sulfoxide (DMSO) are 9:1.
Further, described conditioned medium is in the process of tendon stem cell cultivation, collect the culture fluid containing tendon stem cell secretion thing obtained.
Further, described conditioned medium is prepared by the following method: the tendon stem cell in vegetative period of taking the logarithm, and making cell concentration with cell culture fluid is 1 × 10
5the cell suspension of cell/mL, gets 15mL cell suspension inoculation in diameter 10cm culture dish, in 37 DEG C, 5%CO
2condition under cultivate, the cell culture fluid that every 24h more renews, collect the supernatant of cultivation 24h and/or 48h, centrifugal segregation cell fragment, saves backup in 4 DEG C of constant temperature; Described cell culture fluid is the L-DMEM containing 1%FBS.
Present invention also offers the preparation method of the cryopreserving liquid of a kind of people's heel string source tendon stem cell, comprise the following steps:
(1) cell chulture: tendon stem cell is incubated in the L-DMEM containing 10%FBS, in 37 DEG C, 5%CO
2condition under cultivate 2-3 days, carry out Secondary Culture;
(2) preparation of conditioned medium: the tendon stem cell in vegetative period of taking the logarithm, making cell concentration with cell culture fluid is 1 × 10
5the cell suspension of cell/mL, gets 15mL cell suspension inoculation in diameter 10cm culture dish, in 37 DEG C, 5%CO
2condition under cultivate, every 24h collects supernatant and also changes cell culture fluid, and collect the supernatant of cultivation 24h and/or 48h, centrifugal segregation cell fragment, saves backup in 4 DEG C of constant temperature; Described cell culture fluid is the L-DMEM containing 1%FBS;
(3) according to the volume ratio of 9:1, conditioned medium is mixed with dimethyl sulfoxide (DMSO), the cryopreserving liquid of obtained people's heel string source tendon stem cell.
Further, the ratio of going down to posterity of described Secondary Culture is 1:5.
Further, the centrifugal condition of supernatant is the centrifugal 5min of 2000RPM.
Relative to prior art, beneficial effect of the present invention is: the cryopreserving liquid of people's heel string source of the present invention tendon stem cell is made up of conditioned medium and dimethyl sulfoxide (DMSO), the cost of cell cryopreservation can be reduced on the one hand to a certain extent, the introducing of heterologous material can be avoided on the other hand, compared with regular growth cryopreserving liquid, there is higher clinical safety.Experiment shows, compared with regular growth cryopreserving liquid, the cryopreserving liquid of people's heel string source of the present invention tendon stem cell is less to cellular damage effect in cell cryopreservation process, Cell viability is higher, frozen successful is better than regular growth cryopreserving liquid, may be used for long-term preservation and the application of people's heel string source tendon stem cell.
Accompanying drawing explanation
Fig. 1 is the growth curve chart of people's heel string source tendon stem cell of the frozen recovery afterwards of different cryopreserving liquid.
Embodiment
Describe the present invention below in conjunction with drawings and the specific embodiments.
Embodiment 1, people's heel string source tendon stem cell cryopreserving liquid
Tendon stem cell is incubated in the L-DMEM containing 10%FBS, in 37 DEG C, 5%CO
2condition under cultivate 2-3 days, 1:5 Secondary Culture; The tendon stem cell of taking the logarithm vegetative period, makes cell suspension with cell culture fluid, and adjustment cell concentration is 1 × 10
5cell/mL, gets 15mL cell suspension inoculation in diameter 10cm culture dish, in 37 DEG C, 5%CO
2condition under cultivate, described cell culture fluid is the L-DMEM containing 1%FBS.
When being cultured to 24h, collect supernatant for subsequent use, and the cell culture fluid more renewed continues to cultivate; When being cultured to 48h, again collect supernatant, respectively by the supernatant of twice collection at the centrifugal 5min of 2000RPM, remove cell fragment, save backup in 4 DEG C of constant temperature.By cryopreserving liquid and the regular growth cryopreserving liquid of the freeze-stored cell of formulated shown in table 1.
Table 1 is group cryopreserving liquid composition respectively
The tendon stem cell cryopreserving recovery of embodiment 2, people's heel string source and motility rate
The whole density of cryopreserved human heel string source tendon stem cell is 1-5 × 10
6cell/mL, frozen amount is 1.5mL/ pipe, carries out frozen: first under 4 DEG C of constant temperatures, place 2h according to following program, then places 30min at liquid nitrogen mouth, finally puts into liquid nitrogen (-196 DEG C).
Cell cryopreservation was recovered after 2 weeks, each Group group recovers 3, from liquid nitrogen, take out cryopreservation tube directly put into 37 DEG C of warm water, and shake makes it melt as early as possible at short notice frequently, after thawing in the centrifuge of 720RPM centrifugal 5min, abandon supernatant, in every arm, add 1mL cell culture fluid, count and count to live and average.Concrete, the assay method of cell counting and Cell viability is: 1, trypsinization attached cell, prepares single cell suspension, and does suitably dilution; 2, dye: cell suspension and 0.4% trypan blue solution mix with 9:1 and mix (final concentration is 0.04%); 3, count: in 3min, respectively living cell counting and dead cell; 4, Microscopic observation, dead cell is dyed to obvious blueness, and living cells refuses dye in water white transparency shape.5, cell viability is added up: living cell rate (%)=total viable cell/(total viable cell+dead cell sum) × 100%.The motility rate distribution recorded is as shown in table 2:
Table 2 is group motility rate result respectively
| Group | 0 | 1 | 2 | 3 | 4 | 5 |
| Cell viability (%) | 21.40％ | 98.80％ | 95.20％ | 95.50％ | 93.80％ | 92.30％ |
Cell viability experimental result is as shown in Table 2 known: conditioned medium add the motility rate significantly improving recovery cell.
Multiplication capacity after embodiment 3, cell recovery
Get 12 well culture plates, the cell after recovery is inoculated in hole with the density in 10000cell/ hole by every hole, and every hole adds the L-DMEM liquid 1mL containing 10%FBS, is placed in 37 DEG C, 5%CO
2cultivate in incubator, within every three days, change liquid once.From the 3rd day (cell of just having recovered needs about 24h to conform), plate is taken out every 24h, Stochastic choice 3 hole, suction is abandoned old culture fluid and is cleaned with PBS, add trypsin digestion cell, after stopping digestion, prepare single cell suspension, blow even, get after 10 μ L cell suspensions and 10 μ L0.4% trypan blues mix and be loaded onto on blood cell counting plate, under 10 times of mirrors, count the quantity of tally corner block plaid inner cell, the cell quantity of counting is as shown in table 3, take time as transverse axis, cell number is that the longitudinal axis draws cell growth curve, as shown in Figure 1.
Table 3 different time cell quantity statistical form
Experimental result from shown in table 3 and Fig. 1: the cell proliferative after utilizing the cryopreserving liquid of people's heel string of the present invention source tendon stem cell frozen and conventional cryopreservation recovery no significant difference.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (7)
1. a cryopreserving liquid for people's heel string source tendon stem cell, is made up of conditioned medium and dimethyl sulfoxide (DMSO).
2. the cryopreserving liquid of people's heel string source according to claim 1 tendon stem cell, the volume ratio of described conditioned medium and dimethyl sulfoxide (DMSO) is 9:1.
3. the cryopreserving liquid of people's heel string source according to claim 1 tendon stem cell, described conditioned medium is in the process of tendon stem cell cultivation, collect the culture fluid containing tendon stem cell secretion thing obtained.
4. the cryopreserving liquid of the people's heel string source tendon stem cell according to any one of claim 1-3, described conditioned medium is prepared by the following method: the tendon stem cell in vegetative period of taking the logarithm, and making cell concentration with cell culture fluid is 1 × 10
5the cell suspension of cell/mL, gets 15mL cell suspension inoculation in diameter 10cm culture dish, in 37 DEG C, 5%CO
2condition under cultivate, the cell culture fluid that every 24h more renews, collects the supernatant of cultivation 24h and/or 48h, and centrifugal treating removes cell fragment, saves backup in 4 DEG C of constant temperature; Described cell culture fluid is the L-DMEM containing 1%FBS.
5. a preparation method for the cryopreserving liquid of people's heel string source tendon stem cell, comprises the following steps:
(1) cell chulture: tendon stem cell is incubated in the L-DMEM containing 10%FBS, in 37 DEG C, 5%CO
2condition under cultivate 2-3 days, carry out Secondary Culture;
(2) preparation of conditioned medium: the tendon stem cell in vegetative period of taking the logarithm, making cell concentration with cell culture fluid is 1 × 10
5the cell suspension of cell/mL, gets 15mL cell suspension inoculation in diameter 10cm culture dish, in 37 DEG C, 5%CO
2condition under cultivate, the cell culture fluid that every 24h more renews, collect the supernatant of cultivation 24h and/or 48h, centrifugal segregation cell fragment, saves backup in 4 DEG C of constant temperature; Described cell culture fluid is the L-DMEM containing 1%FBS;
(3) according to the volume ratio of 9:1, conditioned medium is mixed with dimethyl sulfoxide (DMSO), the cryopreserving liquid of obtained people's heel string source tendon stem cell.
6. the preparation method of the cryopreserving liquid of people's heel string source according to claim 5 tendon stem cell, the ratio of going down to posterity of described Secondary Culture is 1:5.
7. the preparation method of the cryopreserving liquid of people's heel string source according to claim 5 tendon stem cell, the centrifugal condition of supernatant is the centrifugal 5min of 2000RPM.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108378019A (en) * | 2018-03-13 | 2018-08-10 | 洛阳轩智生物科技有限公司 | A kind of frozen stock solution of people's stem spermatogonium |
| CN110423722A (en) * | 2019-09-03 | 2019-11-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application and induction method of the tendon stem cell to bone cell differentiation |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5942437A (en) * | 1996-03-12 | 1999-08-24 | University Of South Florida | Method and media for enhancing viability maturation, and cryopreservation of cells |
| CN101486996A (en) * | 2009-02-06 | 2009-07-22 | 浙江大学 | Non-animal source cell blood serum substitute and use thereof |
-
2015
- 2015-10-29 CN CN201510727859.7A patent/CN105211053B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5942437A (en) * | 1996-03-12 | 1999-08-24 | University Of South Florida | Method and media for enhancing viability maturation, and cryopreservation of cells |
| CN101486996A (en) * | 2009-02-06 | 2009-07-22 | 浙江大学 | Non-animal source cell blood serum substitute and use thereof |
Non-Patent Citations (2)
| Title |
|---|
| HAO DANIEL LIN 等: "Human Wharton’s Jelly Stem Cell Conditioned Medium Enhances Freeze-Thaw Survival and Expansion of Cryopreserved CD34+ Cells", 《STEM CELL REV AND REP》 * |
| 朱肖奇等: "组织工程化肌腱种子细胞深低温保存的实验研究", 《生物医学工程学杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108378019A (en) * | 2018-03-13 | 2018-08-10 | 洛阳轩智生物科技有限公司 | A kind of frozen stock solution of people's stem spermatogonium |
| CN108378019B (en) * | 2018-03-13 | 2021-03-02 | 诺赛联合(北京)生物医学科技有限公司 | Cryopreservation liquid for human spermatogonial stem cells |
| CN110423722A (en) * | 2019-09-03 | 2019-11-08 | 广州赛莱拉干细胞科技股份有限公司 | A kind of culture medium and its application and induction method of the tendon stem cell to bone cell differentiation |
| CN111657267A (en) * | 2020-06-17 | 2020-09-15 | 科瑞百奥泰州生物技术有限公司 | Ice-free crystal frozen preservation solution and freezing method for preservation of cartilage, tendon and meniscus |
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