CN100463963C - Freeze preservating method for domestic pig skin tissue - Google Patents
Freeze preservating method for domestic pig skin tissue Download PDFInfo
- Publication number
- CN100463963C CN100463963C CNB2006100969716A CN200610096971A CN100463963C CN 100463963 C CN100463963 C CN 100463963C CN B2006100969716 A CNB2006100969716 A CN B2006100969716A CN 200610096971 A CN200610096971 A CN 200610096971A CN 100463963 C CN100463963 C CN 100463963C
- Authority
- CN
- China
- Prior art keywords
- freeze
- skin tissue
- domestic pig
- liquid nitrogen
- preservating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Abstract
The present invention discloses freeze preserving method for domestic pig ear skin tissue, and features that domestic pig ear skin tissue is first set into freeze protecting liquid at environment temperature 18-24 deg.c to balance for 20-30 min, the freeze protecting liquid is then fumigated with liquid nitrogen vapor to freeze, and the domestic pig ear skin tissue is finally set on the surface of liquid nitrogen for 8-20 before being thrown into liquid nitrogen for preservation. The freeze preserved domestic pig ear skin tissue may be defrosted to restore growth capacity. The present invention is simple in operation, and suitable for long term preservation of somatic cell of rare animal.
Description
Technical field
The present invention relates to fields such as germ plasm resource preservation, transgenosis, the freezing preservation of tissue low-temperature and gene therapy.
Background technology
Somatic cell nuclear transfer technique as produce transgenic animal more efficiently means on breeding and rescue animals on the brink of extinction, receive very big concern.The somatocyte that utilizes the premium animal kind is as the nuclear donor cloned animal, and breeding process is quickened in the restriction of breeding cycle and fertility efficient in can avoiding choosing seeds under the natural condition.Simultaneously, somatic effective long-term frozen is preserved the inconsistent contradiction of supply and demand time that occurs clinically in the time of also can solving nuclear transplantation, and ovary tissue and somatic freezing preservation at present has been widely used in the clinical study and improvement of breed of species such as people, mouse, rabbit, sheep, ox, pig, marmoset and elephant.Usually the chemical substance used as lower molecular weight permeability cryoprotectant is as dimethyl sulfoxide (DMSO) (DMSO), glycerine (GLY), ethylene glycol (EG), propylene glycol (PROH) etc.But they are very big to the adaptability difference of different plant species and different cells.Related organization is in to the research of all kinds of protectant tolerances and preservation effect and yet there are no report on the pig.In addition, engineered being organized in following clinic study has great value, and contained huge economic benefit.Skin tissue engineering also is the focus of research.How making up tissue engineering skin and can set up perfect preservation mechanism to be used for the treatment of disease such as skin injury, is that scientist cuts the problem of capturing solution.The family's pig organ and the mankind make it become the valid model of researching human body organ-tissue at physiology and modal similarity, now existing about fibroblastic vitro culture of pig and the frozen research of inoblast thereof, but the freezing preservation of tame pig skin tissue be yet there are no report.
Summary of the invention
The objective of the invention is to study a kind of skin freezing store method, to set up the somatic novel method of freezing preservation at tame pig.
Technical scheme of the present invention is: freeze preservating method for domestic pig skin tissue; it is characterized in that tame pig ear skin histology is placed frozen solution; be balance 20～30 minutes under 18～24 ℃ of conditions earlier in envrionment temperature; after freezing to frozen solution with liquid nitrogen steam is stifling then; again tame pig ear skin histology is placed on the liquid nitrogen surface after 8～20 hours, drop in the liquid nitrogen and preserve.
Described frozen solution is that concentration is 10%～30% glycerine (GLY).
Described frozen solution is that concentration is 10%～30% dimethyl sulfoxide (DMSO) (DMSO).
Described frozen solution is that concentration is 10%～30% ethylene glycol (EG).
Technology advantages of simple of the present invention can obtain the freezing preservation of tame pig ear skin histology, and the back skin histology that thaws has energy for growth.Adopt the present invention, need not special freezing instrument, sample collection is prior art, and is very convenient, is suitable for the somatic prolonged preservation of rare animal.Tissue block placed DMSO, the GLY of (10%, 20%, 30%) three kinds of concentration gradients and EG pre-equilibration 20～30 minutes before freezing under 18～24 ℃ envrionment temperature, reduce the toxicity of refrigerant and reach freezing before the balance of the inside and outside osmotic pressure of cell.Research also shows, dimethyl sulfoxide (DMSO) concentration be 20% or glycerol concentration be 20% or glycol concentration be 30%, the back tissue block growth result that thaws is best.
Description of drawings
Fig. 1 cultivates 5d for flesh tissue and grows inoblast and form * 40 thereof;
Fig. 2 examines distribution * 40 for Hoechst33342 dyeing microscopic examination tissue block border cell;
Fig. 3 is the cellular form under the light microscopic * 400;
Fig. 4 be under the light microscopic nucleus high-visible * 400;
Fig. 5 is for freezing still strong * 40 of molten back tissue block extended capability;
Fig. 6 is for freezing molten back cellular form, and nucleus is also high-visible * and 400;
Concrete implementing method
1, fresh ear skin histology sample obtains
Select the fewer position of blood vessel on the pig ear tissue, grainer is shaved a mao back alcohol wipe sterilization, gets 1cm with ear forceps or scalpel as far as possible
2～2cm
2The ear tissue of left and right area is put ice chest and is taken back the laboratory rapidly.With scalpel separating table cortex, skin corium and subcutaneous cartilage layers, the skin corium that is separated to cleans 2～3 times in the aseptic PBS of preheating in the super clean bench, and syringe needle is divided into 1mm
3Clean once more behind the fritter 2～3 times.10～20 flesh tissues of picking are affixed on the 25mm that clicks and enters blood plasma in advance at random
2Culturing bottle inwall (blood plasma solidifies at baking oven in advance) is directly cultivated as positive control.Hoechst dyeing is with the identification of cell growing state.
2, skin histology freezing and thawing
Osmotic equilibrium before freezing: subregion drips DMSO, GLY and the EG of (10%, 20%, 30%) three kinds of concentration gradients in the culture dish that siliconizing is crossed; picking 3～5 block organization's pieces immerse respectively in the various frozen solutions at random, and osmotic equilibrium is 20～30 minutes under 18～24 ℃ envrionment temperature.Tissue block after the osmotic equilibrium is added in the freeze pipe of the 1.8mL specification that the different protection of 0.5mL different concns liquid is housed in advance, adopt fast cooling respectively and the two kinds of methods of lowering the temperature are at a slow speed carried out frozen.Cooling at a slow speed: liquid nitrogen steam is refrigerated to protection liquid and freezes, and puts on the liquid nitrogen surface 8～20 hours, and the back is dropped into liquid nitrogen and preserved.Fast cooling: tissue block adds directly input liquid nitrogen preservation in the freeze pipe.
Thaw after preserving 14d, the taking-up freeze pipe rocks in 37 ℃ of water-baths rapidly and thaws, and refrigerating fulid can melt about 1 minute.Tissue block with the fresh preheating DMEM rinsing that contains 10%FCS 2 times, is removed cryoprotectant more earlier with aseptic sucrose liquid rinsing 2 times.
3, the cultivation of freezing back skin histology
Tissue block behind the removal cryoprotectant is attached to six well culture plates interpolation nutrient solution with the EDTA-Ox blood plasma and cultivates.The skin histology nutrient solution consists of: add 10% calf serum, 2% chicken serum, 2mmol/L L-glutaminate, 1mmol/L Sodium.alpha.-ketopropionate, 5.5 * 10 in the DMEM high glucose medium
-5Mol/L beta-mercaptoethanol, 10 μ l/mL non-essential amino acid, 0.2% gentamicin.Hoechst dyeing is with the identification of cell growing state.
Observe every day, the next day change nutrient solution.
The flesh tissue cultivation results shows: cultivate 5d family's pig ear skin histology and begin to grow inoblast.Adherent, the extension of tissue block border cell, rounded or fusiformis distributes on a small quantity.Do Hoechst dyeing evaluation after cultivating 14d, tissues observed piece peripheral cell nuclear is big, intensive and obvious under the fluorescent microscope.Light microscopic is observed inoblast down and is radial or the swirl shape distribution, and draws in the net and caking phenomenon, and cell is fusiformis and sealene triangle or polygon mostly, examines big and clear.More different respectively samples as seen, more little sample ear tissue of age grow fibroblastic time more early and energy for growth strong more, easier expansion.Three the tame pig samples of adopting were respectively about a monthly age, two monthly ages and three monthly ages, and tissue block growth rate is respectively 100%, 100% and 93.3%.Time and energy for growth that tissue block begins to grow cell have individual difference.
The freezing test result shows: for tame pig ear skin histology, the late at least 6d of time ratio flesh tissue that begins to grow after thawing in a organized way.EG and DMSO are higher as protectant survival rate in the quick-freezing method, and GLY is better as protectant refrigerating effect in the slow freezing method.In addition, recovery back tissue block does not all have inoblast and grows under the quick freezing.In a word, the 20%GLY protection is respectively 0 and 82.35% in fast, slow freezing condition undertissue piece growth rate, and the growth rate utmost point under the slow freezing condition is significantly higher than other group and control groups (P＜0.01).
The present invention has compared dimethyl sulfoxide (DMSO) (DMSO), glycerine (GLY), three kinds of lower molecular weight permeabilitys of ethylene glycol (EG) protective material with three kinds of concentration gradients (10%, 20%, 30%) effect to the freezing preservation of tame pig ear skin histology under two kinds of cooling conditions.Prove that with present method saving result it is the most effective to preserving tame pig ear skin histology to make protective material with the GLY of slow freezing and 20%.
Claims (3)
1. freeze preservating method for domestic pig skin tissue; it is characterized in that tame pig ear skin histology is placed frozen solution; be balance 20～30 minutes under 18～24 ℃ of conditions earlier in envrionment temperature; after freezing to frozen solution with liquid nitrogen steam is stifling then; again tame pig ear skin histology is placed on the liquid nitrogen surface after 8～20 hours, drop in the liquid nitrogen and preserve.
2. a kind of freeze preservating method for domestic pig skin tissue according to claim 1 is characterized in that described frozen solution is that concentration is 20% glycerine.
3. a kind of freeze preservating method for domestic pig skin tissue according to claim 1 is characterized in that described frozen solution is that concentration is 30% ethylene glycol.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100969716A CN100463963C (en) | 2006-10-20 | 2006-10-20 | Freeze preservating method for domestic pig skin tissue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100969716A CN100463963C (en) | 2006-10-20 | 2006-10-20 | Freeze preservating method for domestic pig skin tissue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1944636A CN1944636A (en) | 2007-04-11 |
| CN100463963C true CN100463963C (en) | 2009-02-25 |
Family
ID=38044291
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100969716A Expired - Fee Related CN100463963C (en) | 2006-10-20 | 2006-10-20 | Freeze preservating method for domestic pig skin tissue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100463963C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP6817610B2 (en) * | 2016-04-05 | 2021-01-20 | 日本メナード化粧品株式会社 | Three-dimensional cultured epidermis model and its manufacturing method, and how to use the three-dimensional cultured epidermis model |
| CN115152740A (en) * | 2022-06-08 | 2022-10-11 | 四川省畜牧科学研究院 | Long-term pig tissue preservation solution and using method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1040815A (en) * | 1989-05-22 | 1990-03-28 | 中国人民解放军第三○四医院 | H quick freezing liquid and vitrifying store the method for skin |
| CN1460527A (en) * | 2003-07-03 | 2003-12-10 | 中国人民解放军第三○四医院 | Application of selective cell-removed pork skin as skin substitute of human body and its preparation method |
| CN1519314A (en) * | 2003-01-20 | 2004-08-11 | 上海理工大学 | Low temperature method for conserving ture skin obtained from tissue engineering |
-
2006
- 2006-10-20 CN CNB2006100969716A patent/CN100463963C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1040815A (en) * | 1989-05-22 | 1990-03-28 | 中国人民解放军第三○四医院 | H quick freezing liquid and vitrifying store the method for skin |
| CN1519314A (en) * | 2003-01-20 | 2004-08-11 | 上海理工大学 | Low temperature method for conserving ture skin obtained from tissue engineering |
| CN1460527A (en) * | 2003-07-03 | 2003-12-10 | 中国人民解放军第三○四医院 | Application of selective cell-removed pork skin as skin substitute of human body and its preparation method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1944636A (en) | 2007-04-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104145943B (en) | A kind of frozen protection liquid of people's umbilical cord China Tong Shi glue tissue and preparation and application thereof | |
| CN109090100A (en) | A kind of mesenchymal stem cell cryopreserving liquid and preparation method thereof and application method | |
| Wang et al. | Cryopreservation of tissue-engineered dermal replacement in Me2SO: Toxicity study and effects of concentration and cooling rates on cell viability | |
| JP2003530405A (en) | New method for heating frozen samples | |
| CN108617638A (en) | Tissue and/or cell cryopreservation protection liquid and its preparation and application | |
| CN103190393A (en) | Biological sample vitrification carrier and application thereof | |
| CN109526941A (en) | A kind of preservation liquid of fat mesenchymal stem cell | |
| CN104523753A (en) | Preparation method, product and application of human umbilical cord mesenchymal stem cell cultural supernatant active factor and cell lysis buffer | |
| CN109497041A (en) | A kind of cryopreservation methods of adipose tissue | |
| CN102763642B (en) | Cryoprotectant and method for cryopreserving placenta amnion and chorion | |
| CN109619088A (en) | A kind of preservation liquid of fat mesenchymal stem cell | |
| Kusuda et al. | Cryopreservation of chum salmon blastomeres by the straw method | |
| CN100463963C (en) | Freeze preservating method for domestic pig skin tissue | |
| Ge et al. | Skin graft preservation | |
| CN108812642B (en) | Systematic method for preparing placenta tissue according to structural hierarchy and cryopreserving and application | |
| KR20120126634A (en) | Preservation Method for Tissues | |
| CN101884321A (en) | Cryopreservation method for Sinkiang saussurea involucrata cell line | |
| CN102480935B (en) | Method Of Processing Allograft Skin For Transplantation, And Cryopreserved Allograft Skin Produced Thereby | |
| CN108124853A (en) | A kind of store method of mescenchymal stem cell | |
| CN101522890A (en) | Method for freeze preservation of tissue-derived cell | |
| CN1372592A (en) | Carrier for animal cell culture comprising organ tissue slice and animal cell culture method and transplantation method with the use of the carrier | |
| CN106479962B (en) | A kind of embryo in vitro culture solution comprising Cathepsin L inhibitor and its application in freezen protective | |
| CN114946829B (en) | Hair follicle tissue vitrification frozen stock solution | |
| CN108812641A (en) | Human trophoblastic tissue prepares cryopreservation methods and application | |
| CN100567480C (en) | Method for milk cow skin freezing preservation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20090225 Termination date: 20111020 |