CN105211053B - 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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- CN105211053B CN105211053B CN201510727859.7A CN201510727859A CN105211053B CN 105211053 B CN105211053 B CN 105211053B CN 201510727859 A CN201510727859 A CN 201510727859A CN 105211053 B CN105211053 B CN 105211053B
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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, and in particular to a kind of frozen stock solution of people's heel string source tendon stem cell and its
Preparation method.
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 various functioning cells, and medical field is referred to as " general-purpose cell ".Stage of development according to residing for stem cell is divided into embryo
Tire stem cell (embryonic stem cell, ES cell) and adult stem cell (somatic stem cell).According to dry thin
The potentiality of development of born of the same parents is divided three classes：Myeloid-lymphoid stem cell (totipotent stem cell, TSC), multipotential stem cell
(pluripotent stem cell) and unipotent stem cell (unipotent stem cell) (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 adult thighbone is effectively treated
Head necrosis, prevents the further development of disease, protects femoral head, delays the operating time of hip replacement, is the emphasis for the treatment of.
Although the method for current hip-preserving surgery is a lot, such as core decompression, the fibular autograft of vascular pedicle, bone marrow mesenchyma are dry thin
Born of the same parents, surface replacement etc., obtain certain curative effect, but be entirely satisfactory without a kind of technology.Since 2007, domestic and foreign scholars
Tendon stem cell (TDSCs) is separately cultured out from the tendon of humans and animals, the cell has (main point of multi-lineage potential
Turn to Gegenbaur's cell, chondroblast, fat cell) and self-renewal capacity.Tendon stem cell has clear and definite stem cell special
Property, these characteristics are adapted to repair the pathologic process of caput femoris necrosis, therefore are expected to careful as the new kind for the treatment of caput femoris necrosis
Born of the same parents, therefore research people heel string source tendon stem cell important in inhibiting.
Stem cell has potential medical value and application value, and cell preservation technique is to realize these potential values
Basis.Stem cell storage refers to after stem cell is isolated and cultured from different tissues, then by Testing and appraisal,
Then will preserve, in order to be used to stem cell be fed back to patient when clinic needs, reach the purpose for the treatment of disease.Mesh
Preceding conventional cell preservation method has culture and freezes two ways.Wherein culture is preserved, and is taken time and effort, and program is cumbersome, in training
In foster process, particularly long term subculture when, cell be susceptible to variation, cell characteristics lose, lose the value of preservation.
And another cell preservation technique is cell cryopreservation, cell is placed in Cord blood in -196 DEG C of liquid nitrogen using Cryopreservation Technology, can
So that cell is temporarily disengaged from growth conditions and saves its cell characteristics, recovery cell is used for again so when needing
Experiment.And a certain amount of cell is moderately preserved, can prevent because the cell cultivated is contaminated or other accidents
And cell is lost kind, serve the effect of cell conservation.In addition to this it is possible to be bought using the form of cell cryopreservation, posted
Give, exchange and transport some cells.To adding protective agent in culture medium during cell cryopreservation -- the glycerine of final concentration 5%-15% or
Dimethyl sulfoxide (DMSO) (DMSO), can be reduced freezing point of solution, and in addition under the conditions of slow freezing, ICW is appeared, and is reduced
Ice crystal is formed, so as to avoid cellular damage.Method using " slow jelly is melted soon " can preferably ensure cell survival.Standard freezing speed
Degree starts to be -1 to -2 DEG C/min, can accelerate when temperature is less than -25 DEG C, afterwards can be (- 196 in direct plunge into Liquid Nitrogen to -80 DEG C
℃)。
It is typically sub- by hyclone and dimethyl in conventional cells frozen storing liquid or commercially available cells frozen storing liquid at present
The material compositions such as sulfone, but hyclone belongs to heterologous material, complicated component, and there is the wind for introducing pollution and anaphylactogen
Danger, is not suitable for clinical practice.Particularly in cell therapy, the presence of heterologous protein may cause unknown bad
Reaction, has a strong impact on treatment results.
Conditioned medium (condition medium, CM)：Refer to that in cell cultivation process, the cell may be secreted
Active material is in nutrient solution, and wherein cell factor is one of main active material, this to cultivate certain cell or tissue
After, the nutrient solution containing cell secreta is just called conditioned medium.Cell or group can be effectively improved using conditioned medium
The success rate of in vitro culture is knitted, and the conditioned medium of separate sources has different promotions or suppression to different cells, tissue
Effect.
The content of the invention
The present invention is directed to problems of the prior art, there is provided a kind of clinical safety is high, while can protect well again
The cells frozen storing liquid for freezing activity is held, for cryopreserved human heel string source tendon stem cell.
The present invention also provides a kind of method of the frozen stock solution for preparing people's heel string source tendon stem cell.
The present invention is achieved through the following technical solutions the purpose：
A kind of frozen stock solution of people's heel string source tendon stem cell, is made up of conditioned medium and dimethyl sulfoxide (DMSO).
As preferred, the frozen stock solution conditional nutrient solution and dimethyl sulfoxide (DMSO) of people's heel string source tendon stem cell
Volume ratio is 9:1.
Further, the conditioned medium is that being done containing tendon for acquisition is collected during tendon stem cell is cultivated
The nutrient solution of cell secreta.
Further, the conditioned medium is prepared by the following method：Take the logarithm the tendon stem cell in growth period, use
It is 1 × 10 that cell culture fluid is made cell concentration5The cell suspension of cell/mL, takes 15mL cell suspension inoculations in diameter 10cm
Culture dish, in 37 DEG C, 5%CO2Under conditions of cultivate, the cell culture fluid more renewed per 24h collects culture 24h and/or the
The supernatant of 48h, centrifugation removal cell fragment, saves backup in 4 DEG C of constant temperature；The cell culture fluid is the L- containing 1%FBS
DMEM。
Present invention also offers a kind of preparation method of the frozen stock solution of people's heel string source tendon stem cell, including following step
Suddenly：
(1) cell culture：Tendon stem cell is incubated in the L-DMEM containing 10%FBS, in 37 DEG C, 5%CO2Under conditions of
Culture 2-3 days, carries out Secondary Culture；
(2) preparation of conditioned medium：Take the logarithm the tendon stem cell in growth period, cell concentration is made with cell culture fluid
It is 1 × 105The cell suspension of cell/mL, takes 15mL cell suspension inoculations in diameter 10cm culture dishes, in 37 DEG C, 5%CO2Bar
Cultivated under part, supernatant is collected per 24h and cell culture fluid is changed, collect the supernatant of culture 24h and/or 48h, centrifugation is gone
Except cell fragment, saved backup in 4 DEG C of constant temperature；The cell culture fluid is the L-DMEM containing 1%FBS；
(3) according to 9：1 volume ratio mixes conditioned medium with dimethyl sulfoxide (DMSO), people's heel string source tendon is obtained dry thin
The frozen stock solution of born of the same parents.
Further, the passage ratio of the Secondary Culture is 1:5.
Further, the centrifugal condition of supernatant is that 2000RPM is centrifuged 5min.
Relative to prior art, beneficial effects of the present invention are：People's heel string of the invention source tendon stem cell freezes
Liquid is made up of conditioned medium and dimethyl sulfoxide (DMSO), on the one hand can to a certain extent reduce the cost of cell cryopreservation, another
Aspect can avoid the introducing of heterologous material, have clinical safety higher compared with regular growth frozen stock solution.Experiment table
Bright, compared with regular growth frozen stock solution, the frozen stock solution of people's heel string source of the invention tendon stem cell is during cell cryopreservation
Smaller is acted on to cellular damage, Cell viability is higher, freezes effect and is substantially better than regular growth frozen stock solution, can be used for people's heel string
The long-term preservation and application of source tendon stem cell.
Brief description of the drawings
Fig. 1 is the growth curve chart that different frozen stock solutions freeze the people's heel string source tendon stem cell for recovering afterwards.
Specific embodiment
Below in conjunction with drawings and the specific embodiments, the present invention will be described in detail.
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%CO2Under conditions of cultivate 2-3 days, 1:
5 Secondary Cultures；To take the logarithm the tendon stem cell in growth period, be made cell suspension of cell culture fluid, adjustment cell concentration is 1 ×
105Cell/mL, takes 15mL cell suspension inoculations in diameter 10cm culture dishes, in 37 DEG C, 5%CO2Under conditions of cultivate, it is described thin
Born of the same parents' nutrient solution is the L-DMEM containing 1%FBS.
When culture is to 24h, collection supernatant is standby, and the cell culture fluid for more renewing continues to cultivate；When culture extremely
During 48h, supernatant is collected again, the supernatant that will be collected twice respectively is centrifuged 5min in 2000RPM, remove cell fragment,
Saved backup in 4 DEG C of constant temperature.By frozen stock solution and regular growth frozen stock solution that preparation freeze-stored cell is formulated table 1 Suo Shi.
The each group frozen stock solution of table 1 is constituted
Embodiment 2, the tendon stem cell cryopreserving recovery of people's heel string source and motility rate
The whole density of cryopreserved human heel string source tendon stem cell is 1-5 × 106Cell/mL, the amount of freezing is managed for 1.5mL/, is pressed
Frozen according to following procedure：First in 2h is placed under 4 DEG C of constant temperatures, 30min then is placed in liquid nitrogen mouthful, be finally putting into liquid
In nitrogen (- 196 DEG C).
Cell cryopreservation is recovered after 2 weeks, and each Group group recovers 3, cryopreservation tube is taken out from liquid nitrogen and is directly placed into
In 37 DEG C of warm water, and shake makes it melt as early as possible in a short time frequently, and 5min is centrifuged in the centrifuge of 720RPM after thawing,
Supernatant is abandoned, 1mL cell culture fluids are added in each tube, counted and count work and average.Specifically, cell count and cell are lived
The assay method of rate is：1st, pancreatin digestion attached cell, prepares single cell suspension, and make appropriate dilution；2nd, dye：Cell suspension
With 0.4% trypan blue solution with 9:1 mixing mixes (final concentration of 0.04%)；3rd, count：In 3min, difference living cell counting
And dead cell；4th, Microscopic observation, dead cell is dyed to obvious blueness, and living cells refuses dye in colorless and transparent.5th, count thin
Born of the same parents' vigor：Living cell rate (%)=total viable cell/(total viable cell+dead cell sum) × 100%.The motility rate distribution for measuring
As shown in table 2：
The each group motility rate result of table 2
| 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 as shown in Table 2 understands：The addition of conditioned medium significantly improves recovery cell
Motility rate.
Multiplication capacity after embodiment 3, cell recovery
12 well culture plates are taken, the cell after recovery is inoculated in hole with the density in 10000cell/ holes per hole, per hole
The L-DMEM liquid 1mL containing 10%FBS are added, 37 DEG C, 5%CO are placed in2Cultivated in incubator, change liquid once within every three days.From the 3rd day
Start (cell just recovered needs about 24h to adapt to environment), every 24h withdrawing plates, randomly choose 3 holes, old nutrient solution is abandoned in suction
And PBS, plus trypsin digestion cell are used, and after terminating digestion, single cell suspension is prepared, blow even, take 10 μ L cell suspensions and 10 μ
L0.4% trypan blues are loaded onto on blood cell counting plate after mixing, and the number of tally corner block plaid inner cell is counted under 10 times of mirrors
Amount, as shown in table 3, with the time as transverse axis, cell number is that the longitudinal axis draws cell growth curve to the cell quantity of counting, such as Fig. 1 institutes
Show.
The different time cell quantity statistical form of table 3
Experimental result shown in table 3 and Fig. 1：Using the frozen stock solution of people's heel string of the invention source tendon stem cell
Cell proliferative and conventional cryopreservation recovery no significant difference after freezing.
Embodiment described above only expresses several embodiments of the invention, and its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to guarantor of the invention
Shield scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (3)
1. a kind of frozen stock solution of people's heel string source tendon stem cell, is made up of conditioned medium and dimethyl sulfoxide (DMSO)；
The preparation method of the frozen stock solution of people's heel string source tendon stem cell, comprises the following steps：
(1) cell culture：Tendon stem cell is incubated in the L-DMEM containing 10%FBS, in 37 DEG C, 5%CO2Under conditions of cultivate
2-3 days, Secondary Culture is carried out, passage ratio is 1:5；
(2) preparation of conditioned medium：Take the logarithm the tendon stem cell in growth period, be made cell suspension, adjustment cell concentration is 1
×105Cell/mL, takes 15mL cell suspension inoculations in diameter 10cm culture dishes, in 37 DEG C, 5%CO2Under conditions of cultivate, it is described
Nutrient solution is the L-DMEM containing 1%FBS, the cell culture fluid collected the supernatant of culture 24h and more renew, and collects culture
The supernatant of 48h, is centrifuged the supernatant centrifugal treating removal cell fragment collected twice respectively, is saved backup in 4 DEG C of constant temperature；
(3) according to 9：1 volume ratio mixes conditioned medium with dimethyl sulfoxide (DMSO), and people's heel string source tendon stem cell is obtained
Frozen stock solution.
2. people's heel string according to claim 1 is originated the frozen stock solution of tendon stem cell, the conditioned medium and dimethyl
The volume ratio of sulfoxide is 9:1.
3. people's heel string according to claim 1 is originated the frozen stock solution of tendon stem cell, and the conditioned medium is in tendon
Stem cell collects the nutrient solution containing tendon stem cell secretion thing for obtaining during cultivating.
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| CN201510727859.7A CN105211053B (en) | 2015-10-29 | 2015-10-29 | Cryopreservation solution for tendon stem cells derived from human achilles tendon and preparation method thereof |
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| CN105211053B true CN105211053B (en) | 2017-07-07 |
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| 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 |
| CN111657267B (en) * | 2020-06-17 | 2021-02-02 | 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)
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|---|---|---|---|---|
| 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 |
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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 |
|---|
| Human Wharton’s Jelly Stem Cell Conditioned Medium Enhances Freeze-Thaw Survival and Expansion of Cryopreserved CD34+ Cells;Hao Daniel Lin 等;《Stem Cell Rev and Rep》;20130113;第172-183页 * |
| 组织工程化肌腱种子细胞深低温保存的实验研究;朱肖奇等;《生物医学工程学杂志》;20060208;第23卷(第1期);第159-164页 * |
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