CN104222068B - Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium - Google Patents
Low-temperature preserved solid culture medium of skin model and preservation method of low-temperature preserved solid culture medium Download PDFInfo
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- 239000001963 growth medium Substances 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 25
- 238000004321 preservation Methods 0.000 title abstract description 10
- 238000005138 cryopreservation Methods 0.000 claims description 47
- 239000007788 liquid Substances 0.000 claims description 43
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 claims description 39
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims description 36
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- 239000002609 medium Substances 0.000 claims description 28
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract
The invention discloses a low-temperature preserved solid culture medium used for a tissue engineering skin model and a preservation method of the low-temperature preserved solid culture medium. The low-temperature preserved solid culture medium is prepared by the following steps: adding a low-temperature protection agent into a common culture medium for epidermis cells to prepare a basic culture solution; and mixing the basic culture solution with agarose gel and condensing to form the solid culture medium suitable for the skin model. The tissue engineering skin model is embedded into the culture medium, is preserved at 4-8 DEG C for 24-72 hours, and then is resuscitated by a common epidermis cell culture solution. The solid culture medium can be used for reducing tissue activity reduction and structure damages of cells and skin tissues, caused by a low-temperature preservation condition, to the greatest extent.
Description
Technical field
The invention belongs to biomedical sector, it is related to a kind of consolidating of skin model cryopreservation transport
Body culture medium and its store method.
Background technology
Substitute developing rapidly of science with external, skin model is tested as cutaneous safety
Succedaneum, has been widely used in the test of the aspects such as medicine, cosmetics.European substitution method committee
Member's meeting (ecvam) has passed through the checking of two kinds of skin model products of epiderm, episkin
With corresponding test bill.
Because the feature of skin model self performance is it is desirable to its structure and work can be kept in vitro
Property, so that it is guaranteed that the extent of spreading amd application of the accuracy of test result, therefore model is protected by it
Deposit the restriction of traffic condition, also should exclude the impact of xenobiotics simultaneously to greatest extent.Cause
The culture medium that a kind of suitable organization engineering skin model of this exploitation preserves transport is significant.
Store method common at present has cryopreservation and room temperature to preserve two kinds, and cryopreservation may be used again
To be divided into, deepfreeze preserves (4 DEG C～8 DEG C) and freezing preserves (- 80 DEG C and -196 DEG C),
It is also easy to produce ice crystal due to during freezing, and cell, tissue are caused to damage, according to
Freezing protective agent, because protective agent excessive concentration can bring toxic action to model, impact is produced
Product application effect；Therefore, the storage temperature that organization engineering skin model is commonly used is 4 DEG C～8 DEG C,
Conserving time limit is 24h～72h.
However, under 4 DEG C～8 DEG C of cryogenic conditions, especially aseptic in a relatively hypoxia
Under preservation condition, because energy supply system balance is broken, lead to the cell in skin model
Inside and outside ion pumping function is obstructed, and causes intraor extracellular na+/k+When ca2+Concentration is unbalance, cell
The internal enzyme relying on such ion and biochemical reaction process are affected, and lead to cellular physiological events
It is obstructed, cell membrane and cytoskeleton injury, show as cellular swelling, necrosis or apoptosis；In addition,
With the lengthening of holding time, cell also switchs to anaerobic metabolism approach by aerobic metabolism approach, from
And substantial amounts of free radical and acid can be produced, and free radical meeting targeted attack cell membrane system, aggravation
Cell low temperature injury, ultimately causes skin model and loses activity.
For the problems referred to above, in succession occur in that many tissue low-temperatures preserve liquid, such as uw liquid (prestige
The cryopreservation solution that Si Kang star university develops, hts-base (hypothermol),
Hts-frs, l-15 liquid, dmem etc..But these preserve liquid and are mainly used in preserving isolated organ,
And apply such preservation liquid requirement that histoorgan is totally submerged wherein and it needs to certain sets
Standby auxiliary completes to preserve.And skin model is mainly used in chemical compositions in cosmetics medicine
Safety detection, can not contact any chemical substance that may affect test result on its surface,
Therefore above-mentioned store method is inapplicable.
Addition nutrient substance in basic culture solution is mentioned in Chinese patent 200910078302.x
And agar, make solid medium, for preserving organization engineering skin, but this culture medium only limits
Preserve in room temperature, because of many factors such as region, seasons, room temperature preservation condition will be maintained to need spy
Different temperature control device, thus increase considerably cost of transportation；If this culture medium is used for low temperature protect
Deposit transport, under prolonged cold anaerobic condition, cellular energy utilization ways change, by former
The aerobic respiration coming switchs to glycolysiss, and capacity usage ratio is greatly lowered, and needs to supplement in time sugar
Metabolism substrate needed for zymolysis.Simultaneously need to supplement can improve the material of various bioenzyme activities,
For glycolysiss, the substantial amounts of acid producing and free radical also should have antioxidant to be removed, and are somebody's turn to do
Culture medium only comprises to maintain the material of organization engineering skin normal physiological activity from composition,
Do not possess and can meet energy metabolism conversion, the composition of suppression metabolite equivalent damage, thus do not have
Have effect and lower the effect that cold and oxygen deficient damages, consequence is cell because anoxia-induced apoptosis and oxidation are damaged
Wound cannot suppress and lead to whole organizational vitality to decline, after organizational structure is destroyed it is impossible to carry out
Continuous operation.
With people to the continuous research of low temperature injury it was found that much can be used for adjust cell from
Son transhipment, stabilizing cell membrane system and cytoskeleton, remove free radical, improve cellular energy generation
Thank to the chemical substance of the effects such as rate, these materials can play protects cell, tissue to damage from low temperature
The effect of wound, according to the literature, trehalose and sucrose are mainly to maintenance intraor extracellular liquid infiltration
It is pressed with important function；Glucose, adenosine and fructose 1,6-diphosphate can maintain and be organized in aerobic
Energy supply under the conditions of metabolism and anaerobic metabolism is it is ensured that cell normal physiological activity needs；Go
Sideramines (dfo) can effectively reduce cell apoptosis and downright bad ratio after cryopreservation recovery,
Simultaneously for repairing mitochondrial injury, maintain cytoskeletal structure to have good effect, add sweet
Propylhomoserin, the repair to damaged cell of alanine, can effectively reduce cell and tissue
Damage under cold and oxygen deficient state.Alpha-tocopherol (ve) belongs to antioxidant.And vc and
Ve synergism can more effectively play the antioxidation of ve, reduces free radical to cell
Damaging action.
So far still there is no a kind of cryopreservation transport medium of comparatively ideal skin model, this training
Foster base should have minimizing histiocyte cold and oxygen deficient to be damaged, and skin model test result is effectively ensured
Accuracy, simultaneously facilitate operation and long-distance transport feature.
Content of the invention
The defect existing for prior art, the purpose of the present invention is to exist for organization engineering skin
The problems such as reduction of vigor present in cryopreservation transportation, structural damage, provide a kind of solid
The cryopreservation transport medium of body and using method.
The present invention is mainly achieved through the following technical solutions:
This organization engineering skin cryopreservation transport solid culture medium is it is characterised in that its component
Including: cell culture fluid and its adding ingredient.
Described cell culture fluid, refers to mcdb153, dmem, hts liquid, f12, or f12
With dmem mixed-culture medium.Volume ratio 1:1 of f12 and dmem mixed-culture medium or 1:3.
Described adding ingredient includes: buffer system hepes, agarose (low melting gel),
Cryoprotective agent trehalose, sucrose, glycine, alanine, adenosine, alpha-tocopherol, dimension life
Plain c, deferoxamine (dfo), 1,6- fructose diphosphate (fdp).
The concentration of above-mentioned each adding ingredient, be respectively as follows: buffer system hepes 1～100mmol/l,
Agarose 0.25%～2%, trehalose 0.1～10mmol/l, sucrose 0.1～30mmol/l,
Glycine 1～100mmol/l, alanine 0.5～50mmol/l, adenosine 1～100mmol/l,
Alpha-tocopherol 0.01～10mmol/l, vitamin c 0.01～10mmol/l, deferoxamine (dfo)
0.01～10mmol/l, 1,6- fructose diphosphate (fdp) 0.1～10mmol/l.
The compound method of the solid medium of this skin model cryopreservation transport is as follows:
One. preparation a liquid, b liquid
A liquid: in cell culture fluid, be separately added into hepes, trehalose, sucrose, sweet ammonia
Acid, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine (dfo), 1,6- bis- phosphorus
Tart fruit sugar (fdp), adds no sequencing, after fully dissolving, filtration sterilization；
B liquid: by low melting point (28-32 DEG C of plastic, 62-68 DEG C of fusing) agarose deionized water
Dissolving, sterilizing is standby.
Two. vibration mixes
Ratio according to volume ratio 1:1 is slowly added to a liquid in b liquid, after vibration mixes, obtains
Obtain the solid medium of skin model cryopreservation transport.
The using method of the solid medium of skin model cryopreservation transport of the present invention:
1. the skin model that culture completes is embedded solid medium, note together with outside culture cell
Meaning avoids producing bubble to affect preservation effect；
2. solid medium is placed in sealing in sterile bag together with skin model, under the conditions of 4 DEG C～8 DEG C
Preserve 24～72 hours；
3. use in skin model front, tissue engineering model be placed in cultured epidermal cell liquid,
CO2 gas incubator is recovered 18～24 hours, you can for subsequent detection project.
Effective ingredient hepes in the present invention, trehalose, sucrose, glycine, alanine,
Adenosine, alpha-tocopherol, vitamin c, deferoxamine, fructose 1,6-diphosphate etc., are all that low temperature lacks
The good protection agent of cell and tissue under oxygen condition:
First, by adding trehalose and the such macromolecular substances of sucrose, can effectively maintain
Intraor extracellular osmotic pressure is it is ensured that the normal physiological environment of cell；Under the conditions of cold and oxygen deficient, cell
Interior ion transport is abnormal, shows as intracellular power factory-mitochondrial injury, cellular energy
Metabolism cannot be normally carried out, cell membrane swelling etc..Glycine in the present invention and alanine, can
To suppress the conduction of Calcium Signal, to reduce the permeability of swelling cell membrane, prevent ion from turning
The deterioration of fortune abnormal conditions, assistance application deferoxamine repair tissue cell is in cold and oxygen deficient shape simultaneously
The mitochondrial injury causing under state, recovers the supply of cell normal energy and intraor extracellular ion is put down
Weighing apparatus, increases cell membrane stability；Simultaneously because can produce substantial amounts of during cold and oxygen deficient preservation
Free radical and acidic materials, can be further exacerbated by cell membrane damage, by alpha-tocopherol, dimension life
The synergism of plain two kinds of preferable antioxidants of c, and hepes buffer agent set up slow
Rush system, histiocyte can be effectively reduced under cold and oxygen deficient state and in glycolytic cycle
The a large amount of free radicals producing and acid, maintain normal cell growth environment.
Compared with prior art, the present invention passes through to optimize cryopreservation formula of liquid, preferably keeps
Biologic activity after cryopreservation transport of organization engineering skin model and specific function.
Agarose can make culture medium solidify, and be easy to tissue engineering model long-distance transport, be organizational project
The popularization and application of skin model are provided convenience, and have important actual application value.
At present, there are no document or patent report by trehalose, sucrose, phosphofructose, adenosine,
Deferoxamine (dfo), glycine, alanine, vc, the combination application such as alpha-tocopherol is in tissue
In the cryopreservation culture medium of engineering skin.Therefore, the above many kinds of substance of this patent combination application,
Develop a kind of novel solid culture medium, this solid medium can effectively reduce organization engineering skin
Model degree of injury after cryopreservation transport, improves organization engineering skin model and is preserving recovery
Effect afterwards, is mainly reflected in three below aspect:
1. this technology efficiently solves skin model and preserves transportation problem, is keeping skin model biological alive
Property while, be not required to the cryopreservation transport that special temperature control device auxiliary can complete skin model.
2. solid medium preserves and can be prevented effectively from because of other chemical substances of skin model surface contamination
And the inaccurate problem of test result leading to.
3. whole operation process is simple and easy to do, easy to use, and it is convenient to preserve, and recovery is convenient.
Brief description
Fig. 1, the reduced coordinates figure of organization engineering skin model cryopreservation recovery result；
Fig. 2, h＆e coloration result (the 200 times of amplifications) photo for flesh tissue engineering skin；
Fig. 3, for being not added with protective agent and only being preserved with cell culture fluid plus agarose recovering after 72h
Organization engineering skin h＆e coloration result (200 times amplification) photo；
Fig. 4, the h＆e for applying the organization engineering skin of recovery after this inventive method preservation 72h
Coloration result (200 times of amplifications) photo；
Fig. 5, organization engineering skin model hts-frs preserve recovery result.
Specific embodiment
With reference to the accompanying drawings and examples, the present invention will be further described.
Embodiment 1 tissue engineering epidermis skin cryopreservation
Prepared by cryopreservation culture medium: cryopreservation culture medium is with cultured epidermal cell liquid
Based on mcdb153, take 100ml mcdb153 culture medium, by shown final concentration thereto
Add: hepes 100mmol/l, trehalose 0.1mmol/l, sucrose 30mmol/l, sweet ammonia
Sour 50mmol/l, alanine 0.5mmol/l, adenosine 5mmol/l, alpha-tocopherol 1mmol/l,
Vitamin c 0.01mmol/l, deferoxamine (dfo) 10mmol/l, 1,6- fructose diphosphate (fdp)
0.5mmol/l；The agarose solution of 0.5% processing with autoclaving in advance after dissolving is according to 1:1
It is prepared into the solid medium of 0.25% agarose concentration after mixing.
Compound method is as follows:
A liquid: in cell culture fluid, be separately added into hepes, agarose, trehalose, sugarcane
Sugar, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine (dfo),
Fructose 1,6-diphosphate (fdp), adds no sequencing, after fully dissolving, filtration sterilization；
B liquid: agarose (low melting gel) deionized water is dissolved, sterilizing is standby.
It is slowly added to a liquid in b liquid, after vibration mixes, obtain skin model cryopreservation fortune
Defeated solid medium.
Using method is as follows:
By the organization engineering skin successfully constructing together with culture utensil, embedded solid culture primary surface,
After aseptic condition lower sealing, under the conditions of being placed in 4 DEG C, preserve 72h.
Preserve and with the mcdb153 culture fluid of 37 DEG C of pre-temperatures, tissue engineering model is carried out after terminating
Rewarming, carries out subsequent detection after 18h.
Embodiment 2 contains the organization engineering skin cryopreservation of melanocyte
Prepared by cryopreservation culture medium: take f12/dmem (1:1) culture medium 50ml, thereto
Add follow-up Protective substances so as to final concentration is respectively as follows: hepes 1mmol/l, trehalose
10mmol/l, sucrose 0.1mmol/l, glycine 100mmol/l, alanine 50mmol/l,
Adenosine 1mmol/l, alpha-tocopherol 10mmol/l, vitamin c 10mmol/l, deferoxamine (dfo)
0.01mmol/l, 1,6- fructose diphosphate (fdp) 0.1mmol/l；With high pressure in advance after dissolving
The agarose solution of the 2% of sterilization treatment is prepared into 1% agarose concentration according to after 1:1 mixing
Solid medium.
The organization engineering skin containing melanocyte will be successfully constructed together with culture utensil, embedding
Enter solid culture primary surface, after aseptic condition lower sealing, under the conditions of being placed in 4 DEG C, preserve 24h.
Preserve after terminating with f12/dmem (1:3) culture fluid of 37 DEG C of pre-temperatures to organizational project mould
Type carries out rewarming, carries out subsequent detection after 18h.
Embodiment 3 holostrome organization engineering skin cryopreservation
Prepared by cryopreservation culture medium: cryopreservation culture medium is with cultured epidermal cell liquid
F12/dmem (1:3), takes f12/dmem (1:3) culture medium of 50ml, by shown end
Concentration, is added to: hepes 50mmol/l, trehalose 5mmol/l, sucrose
10mmol/l, glycine 1mmol/l, alanine 10mmol/l, adenosine 50mmol/l, α-
Tocopherol 0.01mmol/l, vitamin c 5mmol/l, deferoxamine (dfo) 2.5mmol/l,
1,6- fructose diphosphate (fdp) 10mmol/l；3% processing with autoclaving in advance after dissolving
Agarose solution be prepared into the solid medium of 1.5% agarose concentration according to after 1:1 mixing.
Compound method is as follows:
A liquid: in cell culture fluid, be separately added into hepes, agarose, trehalose, sugarcane
Sugar, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine (dfo),
Fructose 1,6-diphosphate (fdp), adds no sequencing, after fully dissolving, filtration sterilization；
B liquid: agarose (low melting gel) deionized water is dissolved, sterilizing is standby.
It is slowly added to a liquid in b liquid, after vibration mixes, obtain skin model cryopreservation fortune
Defeated solid medium.
Using method is as follows:
By the holostrome successfully constructing organization engineering skin together with culture utensil, embedded solid medium
Surface, after aseptic condition lower sealing, preserves 72h under the conditions of being placed in 4 DEG C.
Preserve and with the dmem culture fluid of 37 DEG C of pre-temperatures, tissue engineering model is carried out again after terminating
Temperature, carries out subsequent detection after 18h.
Embodiment 4 holostrome organization engineering skin cryopreservation
Prepared by cryopreservation culture medium: cryopreservation culture medium is with cultured epidermal cell liquid
Based on f12/dmem (1:1), take 50ml f12/dmem (1:1) culture medium, by shown
Final concentration, is added to: hepes 25mmol/l, trehalose 2.5mmol/l, sucrose
15mmol/l, glycine 30mmol/l, alanine 15mmol/l, adenosine 100mmol/l,
Alpha-tocopherol 5mmol/l, vitamin c 5mmol/l, deferoxamine (dfo) 5mmol/l, 1,6-
Fructose diphosphate (fdp) 2.5mmol/l；0.5% processing with autoclaving in advance after dissolving
Agarose solution be prepared into the solid medium of 0.25% agarose concentration according to after 1:1 mixing.
Compound method is as follows:
A liquid: in cell culture fluid, be separately added into hepes, agarose, trehalose, sugarcane
Sugar, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine (dfo),
Fructose 1,6-diphosphate (fdp), adds no sequencing, after fully dissolving, filtration sterilization；
B liquid: agarose (low melting gel) deionized water is dissolved, sterilizing is standby.
It is slowly added to a liquid in b liquid, after vibration mixes, obtain skin model cryopreservation fortune
Defeated solid medium.
Using method is as follows:
By the holostrome successfully constructing organization engineering skin together with culture utensil, embedded solid medium
Surface, after aseptic condition lower sealing, preserves 72h under the conditions of being placed in 4 DEG C.
Preserve and with the mcdb153 culture fluid of 37 DEG C of pre-temperatures, tissue engineering model is carried out after terminating
Rewarming, carries out subsequent detection after 18h.
Embodiment 5 holostrome organization engineering skin cryopreservation
Prepared by cryopreservation culture medium: cryopreservation culture medium is with cultured epidermal cell liquid
Based on mcdb153, take 50ml mcdb153 culture medium, by shown final concentration, Xiang Qi
Middle interpolation: hepes 15mmol/l, trehalose 7.5mmol/l, sucrose 0.1mmol/l, sweet
Propylhomoserin 25mmol/l, alanine 50mmol/l, adenosine 50mmol/l, alpha-tocopherol 5mmol/l,
Vitamin c 5mmol/l, deferoxamine (dfo) 1mmol/l, 1,6- fructose diphosphate (fdp)
10mmol/l；The agarose solution of 2% processing with autoclaving in advance after dissolving is according to 1:1
It is prepared into the solid medium of 1% agarose concentration after mixing.
Compound method is as follows:
A liquid: in cell culture fluid, be separately added into hepes, agarose, trehalose, sugarcane
Sugar, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine (dfo),
Fructose 1,6-diphosphate (fdp), adds no sequencing, after fully dissolving, filtration sterilization；
B liquid: agarose (low melting gel) deionized water is dissolved, sterilizing is standby.
It is slowly added to a liquid in b liquid, after vibration mixes, obtain skin model cryopreservation fortune
Defeated solid medium.
Using method is as follows:
By the holostrome successfully constructing organization engineering skin together with culture utensil, embedded solid medium
Surface, after aseptic condition lower sealing, preserves 72h under the conditions of being placed in 4 DEG C.
Preserve and with the mcdb153 culture fluid of 37 DEG C of pre-temperatures, tissue engineering model is carried out after terminating
Rewarming, carries out subsequent detection after 18h.
The following is the experiment test situation of above-described embodiment:
Fig. 1, the reduced coordinates figure of organization engineering skin model cryopreservation recovery result
Be with solid medium shown in the present invention organization engineering skin model is carried out low temperature (4～
8 DEG C) preserve and use the relative activity after common epidermal cell culture base recovery, wherein normal control is new
The organization engineering skin group of preparation, 1 is to be not added with protective agent group, and 2 is to add each protective agent group,
What t molded line represented is the standard deviation of experiment.
The result shows, adds protective agent group 2 and is relatively not added with organizing 1 after preservation and recovery,
Organization engineering skin vigor keeps preferably it was demonstrated that adding protective agent group can effectively reduce organizational project
Skin model Cryopreservation Injury, improves recovery vigor after organization engineering skin cryopreservation.
The h＆e coloration result that Fig. 2, Fig. 3 are respectively flesh tissue engineering skin (is put for 200 times
Greatly) photo only preserves recovery after 72h with cell culture fluid plus agarose with being not added with protective agent
H＆e coloration result (the 200 times of amplifications) photo of organization engineering skin.
By diagram as can be seen that flesh tissue result is well arranged, cell quantity is many, form
Completely, and after cryopreservation organization engineering skin occur organizational structure lack unity and coherence clear, carefully
Born of the same parents' number reduces, and cell shrinkage and cavity, illustrates that cryopreservation exists to organization engineering skin
More serious damaging action.
Fig. 4 is the h＆e of the organization engineering skin applying this inventive method to recover after preserving 72h
Coloration result (200 times of amplifications) photo.
Fig. 5 is the h＆e dye that application hts-frs preserves recovery after 72h to organization engineering skin
Color result (200 times of amplifications) photo；
Compared as can be seen that being not added with protective agent group and business-like hts-frs by Fig. 4, Fig. 5
Culture medium all cannot be resisted the organization engineering skin causing under cold and oxygen deficient state and damage, and leads to
Vigor declines and organizational structure is damaged.The group after method cryopreservation 72h in the application present invention
Knit engineering skin in organizational vitality, cellular morphology and organizational structure all with freshly prepd group of weaver
Journey skin no significant difference.
Claims (3)
1. a kind of cryopreservation solid medium that can be used for organization engineering skin model, its component includes: cell culture fluid and its adding ingredient, cell culture fluid is mcdb153, dmem, hts liquid, f12, f12/dmem；Volume ratio 1:1 of f12 and dmem mixed-culture medium or 1:3；It is characterized in that, adding ingredient includes: buffer system hepes, agarose
Low melting gel, cryoprotective agent trehalose, sucrose, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine and fructose 1,6-diphosphate；
Described each adding ingredient, concentration is respectively as follows: buffer system hepes 1～100mmol/l, agarose 0.25%～2%, trehalose 0.1～10mmol/l, sucrose 0.1～30mmol/l, glycine 1～100mmol/l, alanine 0.5～50mmol/l, adenosine 1～100mmol/l, alpha-tocopherol 0.01～10mmol/l, vitamin c 0.01～10mmol/l, deferoxamine (dfo) 0.01～10mmol/l, 1,6- fructose diphosphate (fdp) 0.1～10mmol/l.
2. a kind of compound method of the cryopreservation solid medium that can be used for organization engineering skin model as claimed in claim 1 it is characterised in that:
One. preparation a liquid, b liquid
A liquid: in cell culture fluid, it is separately added into hepes, trehalose, sucrose, glycine, alanine, adenosine, alpha-tocopherol, vitamin c, deferoxamine and fructose 1,6-diphosphate, after fully dissolving, filtration sterilization；
B liquid: the agarose of 28-32 DEG C of plastic dissolves in 62-68 DEG C of deionized water, sterilizing is standby；
Two. vibration mixes
According to the ratio of volume ratio 1:1, b liquid is slowly added to a liquid, after vibration mixes, obtains the solid medium of skin model cryopreservation transport.
3. a kind of using method of the cryopreservation solid medium that can be used for organization engineering skin model as claimed in claim 1 it is characterised in that:
1) skin model that culture completes is embedded solid medium together with outside culture cell；
2) solid medium is placed in sealing in sterile bag together with skin model, preserves 24～72 hours under the conditions of 4 DEG C～8 DEG C；
3) in skin model using front, tissue engineering model is placed in cultured epidermal cell liquid, recovers 18～24 hours in CO2 gas incubator, you can for subsequent detection project.
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| CN105230608B (en) * | 2015-10-23 | 2023-05-09 | 陕西艾尔肤组织工程有限公司 | Preservation method of tissue engineering skin and tissue engineering skin |
| CN107164306B (en) * | 2017-06-18 | 2020-09-08 | 广东博溪生物科技有限公司 | Solid culture medium for cornea model low-temperature preservation and preparation method and application method thereof |
| CN109699631B (en) * | 2018-12-21 | 2021-07-16 | 广州悦清再生医学科技有限公司 | Biological material semi-solid storage medium and application |
| CN111100838B (en) * | 2019-12-27 | 2023-05-09 | 广东博溪生物科技有限公司 | Low-temperature preservation and transportation culture medium |
| CN111631212B (en) * | 2020-06-28 | 2021-12-24 | 济南磐升生物技术有限公司 | Low-temperature storage method of multifunctional 3D recombinant skin model |
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