CN102791278A - Cell protectants comprising placenta extracts - Google Patents
Cell protectants comprising placenta extracts Download PDFInfo
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- CN102791278A CN102791278A CN2010800646536A CN201080064653A CN102791278A CN 102791278 A CN102791278 A CN 102791278A CN 2010800646536 A CN2010800646536 A CN 2010800646536A CN 201080064653 A CN201080064653 A CN 201080064653A CN 102791278 A CN102791278 A CN 102791278A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
Abstract
Disclosed is a cell protectant comprising a placenta extract, more specifically a cell protectant for storing and protecting animal cells, including amino acids, minerals, vitamins, growth factors and placenta extract.
Description
Technical field
The present invention relates to comprise the cytoprotective of intacellin, more specifically, relate to and be used to store and the cytoprotective of the cell that watches for animals that said cytoprotective comprises aminoacid, mineral, vitamin, somatomedin and intacellin.
Background technology
Usually, cell is a freezing under-196 ℃ extremely low temperature, and the acquisition of when the needs living cells, thawing rapidly.Although the survival rate of frozen-thawed cell (frozen-and-then-thawed cells) is according to cell type or operator's proficiency level and difference, normal and useful cell, non-cancer cell, survival rate very low.In addition, only needing transportation or storing several hours or several days, and be not under the situation of long period transportation or storage, cryopreservation (cryopreservation) method is unpractiaca.
Recently, developing various diseases is treated in use from isolating mature cell of animal tissue or stem cell cell therapy technology.Therefore, need develop the viability (viability) that to keep the mature cell that obtains from tissue and stem cell for a long time and the protective agent that better preservation is provided strongly.At present, the culture medium that temporarily will be used for culture of animal cells is as cytoprotective, and about useful protectant research of being exclusively used in the preservation cell seldom.
Cultivate to human stem cell, use the culture medium that comprises animal serum (hyclone).Even owing to use the hyclone cultured cells after with serum-free solution washing cell, still the risk with infection bovine spongiform encephalopathy or other diseases is relevant, so the U.S. forbids use hyclone cultured cells is transplanted to the mankind.From people's bone marrow and from fatty tissue the technology of separating mesenchymal stem cell (mesenchymal stem cell) be disclosed in Pittenger et al. respectively [Science 284: 143-147; 1997] and Van et al. [J.Clin.Invest.58: 699-704; 1976], wherein use α-MEM or DMEM and 10-20% hyclone to come cultured cell.Yet the use of animal serum (hyclone) is relating to having dispute aspect human implantation's the safety.
In order to overcome this problem, [Transplantation.2000Dec 27 for Kuznetsov SA, et al. to attempt end user's serum replacement hyclone; 70 (12): 1780-7].Yet when not carrying out any processing when end user's serum in the cell culture medium, stem cell is lost their characteristic easily, and for example propagation and differentiation capability reduce, are easy to be divided into osteocyte or the like.In addition, after the transplanting, the viability of stem cell is extremely low and can not bring into play their ability fully.In order to optimize the tissue regeneration of adult stem cell, need study viability and optimization that improves the stem cell of transplanting and the technology that improves transplantation effect.
Summary of the invention
Technical problem
Therefore, the purpose of this invention is to provide a kind of cytoprotective, said cytoprotective can improve the viability of the cell that stores at low temperatures than existing Zooblast culture medium, and can improve propagation and the ability (capability) of stem cell.
Technical scheme
Inventors of the present invention are devoted to solve aforementioned prior art problems.The result; They have developed a kind of cytoprotective that comprises aminoacid, mineral, vitamin, somatomedin and intacellin, and confirm this cytoprotective not use animal serum and improve the viability of zooblast and can strengthen the ability of stem cell.
Beneficial effect
Cytoprotective of the present invention can be used for storing safely cell, is not only stem cell and also has various other zooblasts.When the cell that is used for cell therapy in low temperature storage and when transportation, this cytoprotective can be used for protecting said cell.When before cultivation, handling zooblast with cytoprotective of the present invention, cell proliferation rate that can obtain to improve and stem cell ability.Therefore, cytoprotective of the present invention is very useful as the protective agent of preservation cell and raising stem cell ability.
Description of drawings
Through below in conjunction with the description of accompanying drawing to the preferred implementation that provides, above-mentioned and other purposes of the present invention, feature and advantage will be obvious, in the accompanying drawings:
Fig. 1 compared control medium or in CeLLGUARD solution of the present invention low temperature (4 ℃) stored 72 hours the cell survival rate of isolating adipose cell when not cultivating from fatty tissue;
Fig. 2 a has compared the stem cell ability of the mescenchymal stem cell of handling with control medium or CeLLGUARD that is derived from fatty tissue;
Fig. 2 b has compared the cell proliferation rate of the mescenchymal stem cell of handling with control medium or CeLLGUARD that is derived from fatty tissue;
Fig. 3 a has compared the mescenchymal stem cell that is derived from fatty tissue handled with control medium or the CeLLGUARD cell proliferation rate 7 days, 10 days and 14 days;
Fig. 3 b shows with control medium or CeLLGUARD process source behind the mescenchymal stem cell of fatty tissue, identifies the result of surface expression labelling;
Fig. 4 a and Fig. 4 b have compared the effect of the mescenchymal stem cell healing of wound of handling with control medium or CeLLGUARD;
Fig. 5 shows after handling from skin histology isolating keratinocyte (keratinocyte) with control medium or CeLLGUARD and cultivating, and identifies the result of surface expression labelling; And
Fig. 6 has compared from the isolating keratinocyte of skin histology after handling with control medium or CeLLGUARD, the stem cell ability after the cultivation.
Preferred forms
Hereinafter, will be described in detail with reference to the attached drawings embodiment of the present invention.
On the one hand, the present invention provides a kind of cytoprotective that comprises aminoacid, mineral, vitamin, somatomedin and intacellin.
In the present invention, cytoprotective refers to a kind of culture medium composition, and said composition is used under low temperature or room temperature, storing zooblast several hours to several days, is used to experiment, operation or processing until said zooblast.Cytoprotective with in order to grow and to breed the conventional animal cell culture medium that specific cells optimizes different in composition, content and use.Although cytoprotective of the present invention can be used as the additive of cultured cell and adds, it self is not enough to as culture medium.Also promptly, for the cultivation and the growth of specific cells, recommend to use the culture medium of optimizing to them, and do not recommend to use cytoprotective of the present invention self as culture medium.Said cytoprotective can be used for the preservation cell, adds in the culture medium to improve the viability or the growth of cell, perhaps in abundant culture medium, handles cell before the growth at cell.
Cytoprotective of the present invention can be used for zooblast, preferred stem cell, more preferably adult stem cell.Usually, zooblast needs aminoacid, mineral, vitamin etc. so that survival.Normally used Zooblast culture medium comprises these compositions.Although cytoprotective of the present invention also comprises those compositions such as aminoacid, mineral, vitamin etc.; But the composition of these compositions in the cytoprotective is different with Zooblast culture medium with content, because the purpose of cytoprotective various zooblasts that are preservations rather than cultivation or proliferative cell (referring to table 1-4).
In the embodiment of the invention 1, adipose cell is carried out the cell survival property testing.When cell stores 72 hours at low temperatures, obtained at least 70% viability (referring to table 1).In addition, the test result of the cell proliferation rate of keratinocyte and fat-derived stem cells and stem cell ability also significantly is superior to the test result (referring to Fig. 2 and Fig. 6) of existing Zooblast culture medium.
Existing Zooblast culture medium comprises that animal serum is as nutritional labeling.Although this helps cytotostatic growth, when comprising under animal serum and room temperature or the low temperature (non-ultralow temperature) placement for a long time in the cytoprotective, can cause the reduction of cell survival property.Yet; Because cytoprotective of the present invention is not to be used for cultivating or proliferative cell; Be used to experiment or operation but be used to improve cell survival property until this cell, therefore cytoprotective of the present invention be serum-free and contain the required composition of promising greater amount survival.
Especially, cytoprotective of the present invention can serviceably be used for the preservation stem cell.For a lot of cell therapy of recent research, cytoprotective of the present invention can be used as the protective agent that can store and transport stem cell.In addition, through handling stem cell, can improve the ability of stem cell with cytoprotective of the present invention.When handling stem cell, cytoprotective of the present invention has reduced cell death and activation cell.Therefore, cytoprotective of the present invention is very useful for stablizing stem cell.
Preferably, in cytoprotective of the present invention, except 20 kinds of constitutive protein matter the primary amino acid, aminoacid also comprises L-cystine and L-hydroxyproline (L-Hydroproline).
The L-hydroxyproline is used to resist the oxidation attack of reductive DNA, repairs damaged dna and also produces collagen protein.It as to the collagen protein biosynthesis with keep essential amino acids---hydroxyproline (hydroxyproline) and hydroxylysine provide the donor of hydroxyl, be the necessary cofactor in the collagen protein biosynthesis.Therefore, estimate the L-hydroxyproline play antioxidant be used for remove the reactive oxygen species and prevent cell death.
The L-cystine contains sulfur and is the component of hair or angle (horn).Especially, the L-cystine is present in the keratin with about 10% amount, and is easy to be reduced into the L-cysteine.The L-cystine plays an important role in the activity of the tertiary structure that determines polypeptide and enzyme or hormone.Therefore, the L-cystine plays an important role for cytoactive.
Preferably, in cytoprotective of the present invention, except the required general element such as calcium, sodium, potassium, copper, ferrum, magnesium and zinc of cell growth, mineral also comprises trace element zirconium, germanium and vanadium.The mineral that comprises in the cytoprotective of the present invention can exist with the form of inorganic salt, for example ZrOCl
28H
2O, GeO
2And NaVO
3
Preferably, in cytoprotective of the present invention, vitamin comprise can inhibitory reaction property oxygen species calcium pantothenate, choline chloride, folic acid, inositol and ascorbic acid.
Minerals and vitamins is used to remove the toxicant of cell (for example antioxidation) generation and promote cellular metabolism, improves cell survival property thus.These functions are considered to obtain through the interaction between the composition that comprises in the cytoprotective of the present invention.
Preferably, in cytoprotective of the present invention, somatomedin comprises basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).These somatomedin are that cell survival is necessary and also influence the activation of cell.Except somatomedin, cytoprotective of the present invention can further comprise serum substitute, for example albumin.
Preferably, the aforementioned composition of cytoprotective exists in following content: based on the whole protecting agent, aminoacid 0.5 is to 20g/L, and vitamin 0.01 is to 1.0g/L, and mineral 1.0 to 100g/L and somatomedin 0.001 are to 0.1mg/L.
In cytoprotective of the present invention, include intacellin, keeping cell survival at memory period, and can also between the culture period after the storage, improve cytoactive if desired.Can be intacellin or Placenta Hominis hydrolyzate.Based on protectant gross weight meter, intacellin or Placenta Hominis hydrolyzate can be with 1-20wt%, and the amount of preferred 5-20wt% is included in the cytoprotective.
Intacellin can be through preparing with the Placenta Hominis of hydrochloric acid hydrolysis from the healthy subjects that hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are negative.Particularly, in order to make virus or analog inactivation, surpassing 101 ℃ of processing Placenta Hominiss at least 1 hour, sterilized in 60 minutes through about 121 ℃ autoclaved then with hydrochloric acid.The Placenta Hominis hydrolyzate can be through preparing to remove fat and to carry out abundant hydrolysis with pepsin and hydrochloric acid with the acetone treatment Placenta Hominis.In an embodiment of the present invention, use from commercially available intacellin of buying of Hwa Sung Bio Pharm and Placenta Hominis hydrolyzate.
In the preferred embodiment of the present invention, cytoprotective comprises the composition of listing among the table 1-4.The cytoprotective that inventors of the present invention develop is known as " CeLLGUARD ", component and content list among the table 1-4.
Cytoprotective of the present invention can be used for improving under 0-37 ℃, preferably under 0-10 ℃ low temperature, and more preferably under 0-4 ℃ low temperature, most preferably under 4 ℃ low temperature, the viability of the cell of storage.
Be different from the existing stem cell media that is used for cell proliferation and differentiation, cytoprotective of the present invention has improved ability of stem cell and the cell proliferation rate of stem cell, and does not change cell characteristics.When handling stem cell with cytoprotective of the present invention when cultivating then, stem cells hyperplasia is good and have a stem cell ability of raising.In embodiments of the invention 2 and 3, measured the stem cell ability and the cell proliferation rate of fat-derived mescenchymal stem cell.The result discloses, and than existing stem cell media, cytoprotective of the present invention has significant effect.
Cytoprotective of the present invention can be used for storing and the transport cells therapeutic agent, to strengthen the curative effect of cellular therapeutic agent.Term " cellular therapeutic agent " refers to a kind ofly to be used to treat, the new ideas medicine of diagnosis or prevent disease purpose; Said medicine passes through in-vitro multiplication or screens from body, allochthonous or xenogeneic living cells; Perhaps otherwise change the biological nature of cell, to recover or to improve the function of said cell and tissue.Particularly, the somatic cell of patient self or other animals can be bred, and perhaps stem cell can be divided into the cell of wanting, with the treatment burn or such as the various diseases of not healing of cancer, dementia (dementia) etc.In this respect, the U.S. is being carried out to hundred kinds of clinical experiments, and Korea S is also actively being studied.
Before the cell that is used for cell therapy is used, store safely and transport that they are extremely important.Most zooblast, particularly stem cell are separating the just dead or inactivation in back from their tissue.In embodiments of the invention 1, use existing adipose cell culture medium or cytoprotective of the present invention, will under 4 ℃ low temperature, store 72 hours from the isolating adipose cell of fatty tissue, measure cell survival property then.The cell that uses cytoprotective of the present invention to store demonstrates 70% or higher cell survival rate, and the cell that uses existing adipose cell culture medium to store demonstrates 50% or lower cell survival rate.Therefore, cytoprotective of the present invention keeps cell survival very useful before being used for cell therapy for cell.
The invention embodiment
Now embodiment and experiment will be described.Following embodiment and experiment are only used for illustrative purposes, and are not intended to limit the scope of present disclosure.
Preparation example 1: the preparation of cytoprotective
Other components that aminoacid through table 1 is listed, the mineral that table 2 is listed, vitamin that table 3 is listed and table 4 are listed are mixed, and prepare cytoprotective of the present invention, and called after " CeLLGUARD ".As contrast, fat-derived mescenchymal stem cell is used the DMEM/F12 that further contains 10% hyclone (1:1) culture medium of Invitrogen, and keratinocyte is used the KGM culture medium of Lonza.
[table 1] aminoacid
[table 2] mineral
[table 3] vitamin
[table 4] other components
Embodiment 1: the comparison of adipose cell viability
Will be from fatty tissue isolating 1 * 10
6Individual adipose cell is put into the bottle that contains control medium and cytoprotective of the present invention (hereinafter is called " CeLLGUARD ") respectively.After 4 ℃ of placements, viable count is counted, to measure the viability of cell with predetermined time interval.Cell counting uses blood cell calculator to carry out at microscopically after being to dye with trypan blue (trypan blue) pair cell.
Fig. 1 compared control medium or in CeLLGUARD solution low temperature (4 ℃) stored 72 hours from the cell survival rate of the isolating adipose cell of fatty tissue when not cultivating.Through viable count being measured the viability of cell.Like Fig. 1 finding, the cell survival rate of the adipose cell that in CeLLGUARD, stores is 72%, and the cell survival rate that in the adipose cell culture medium, stores is merely 45%.Also promptly, than control medium, CeLLGUARD makes cell survival rate increase by 60%.
Embodiment 2: the affirmation of the stem cell ability of fat-derived mescenchymal stem cell
1) CFU analyzes to confirm the stem cell ability
, and get 500 cell inoculations back on 6 orifice plates and cultivated 7 days from the mescenchymal stem cell of fatty tissue 1 hour with control medium or CeLLGUARD process source.In order to measure colony forming unit (CFU, colony-forming units), with taking out fully in the culture medium slave plate, the 1:1 mixture solution pair cell dyeing that adds Luo Danhong and Nile blue is after 30 minutes, to the colony counting number.
Fig. 2 a has compared the stem cell ability of the mescenchymal stem cell that is derived from fatty tissue.From the cell of fatty tissue 1 hour, confirm the stem cell ability through measuring CFU with control medium or CeLLGUARD process source then.Like Fig. 2 a finding, CeLLGUARD has obtained 10% colony formation efficient (CFE), and this is more than 3 times of control medium (about 3%).
2) confirm cell proliferation rate
With control medium or CeLLGUARD process source from the mescenchymal stem cell of fatty tissue 1 hour, and with per unit area (cm
2) have 25 cells on culture plate, to inoculate back cultivation 7 days.Collecting cell with trypan blue dyeing, and uses blood cell calculator to count at microscopically.
Fig. 2 b has compared from the cell proliferation rate of the isolating mescenchymal stem cell of fatty tissue.As scheme finding, the cell number that CeLLGUARD obtains is 1.5 * 10
5, this comparison is according to culture medium (9.5 * 10
4) many about 60%.
Embodiment 3: the affirmation of the cell proliferation rate of fat-derived mescenchymal stem cell and surface expression labelling
Evaluation
1) cell counting
Handle from the isolating mescenchymal stem cell of fatty tissue 1 hour with control medium or CeLLGUARD, and get 1 * 10
5Individual cell inoculation is on culture plate.Cultivate after 7 days, 10 days and 14 days, collecting cell with trypan blue dyeing, and uses blood cell calculator to count at microscopically.
Fig. 3 a has compared the cell proliferation rate of the fat-derived mescenchymal stem cell of handling with control medium or CeLLGUARD.Like Fig. 3 a finding, behind the cell culture after handling 1 hour with CeLLGUARD, cell number increases continuously, and reaches 40,000 at the 7th day, reaches 115,000 on the 10th day, and reaches about 180,000 at the 14th day.Especially, the 14th day cell number than the cell number (about 100,000) of the cell of handling with control medium many about 80%.
2) flow cytometer
Handle from fatty tissue isolated cells 1 hour with control medium or CeLLGUARD, and be seeded on the culture plate.Cultivate after 7 days; Collecting cell with phosphate buffer (PBS) thorough washing, washs with PBA (1% bovine serum albumin in PBS) again; And with primary antibody (the Santa Cruz of stem cell labeling CD29; USA), (Millipore, USA) (Millipore USA) handled 1 hour with 1:100 with the primary antibody of CD105 for the primary antibody of CD34.Use PBA thorough washing cell 2 times then, with FITC-put together two anti-(Jackson ImmunoResearch Laboratory USA) handled 30 minutes, and (BD USA) analyzes to use CellQuest Pro software then.
Fig. 3 b shows with control medium or CeLLGUARD process source behind the mescenchymal stem cell of fatty tissue, identifies the result of surface expression labelling.Carry out flow cytometry analysis with identification of cell surface expression labelling.As a result, the cell of handling with CeLLGUARD does not demonstrate the obvious change of cell surface marker, and this discloses that CeLLGUARD can promote the cell growth and the basic characteristic that do not change cell.
Embodiment 4: use the affirmation of animal model to the stem cell ability
Anesthesia CF1 mice also uses razor to remove the back hair neatly.After using the 8mm card punch to make wound, with 1 * 10
6Handle with control medium or CeLLGUARD and be sprayed on the wound from the isolating mescenchymal stem cell of fatty tissue after 1 hour.The wound healing degree is observed in the drug of topical application after 6 days.
Fig. 4 a and Fig. 4 b have compared the effect of the healing of wound of the mescenchymal stem cell of handling with control medium or CeLLGUARD.Fig. 4 a illustrates the result who observed wound healing after the drug of topical application in 6 days, and Fig. 4 b illustrates the result who measures wound size.As scheme finding, the mescenchymal stem cell of handling with CeLLGUARD has caused faster and better wound healing than the mescenchymal stem cell of handling with control medium.
Embodiment 5: the affirmation of the surface expression labelling of keratinocyte
Handle from the keratinocyte of application on human skin separate tissue 1 hour with control medium or CeLLGUARD.After the cultivation; Collecting cell is used the PBS thorough washing, washs again with PBA (1% bovine serum albumin in PBS); Use the primary antibody of keratinocyte labelling CD24 and primary antibody (the Santa Cruz of stem cell labeling CD29 then; USA), (Millipore, USA) (Millipore USA) handled 1 hour with 1:100 with the primary antibody of CD90 for the primary antibody of CD34.Use PBA thorough washing cell 2 times then, with FITC-put together two anti-(Jackson ImmunoResearch Laboratory USA) handled 30 minutes, and (BD USA) analyzes to use CellQuest Pro software then.
Fig. 5 illustrates the result of the surface expression labelling of identifying keratinocyte.Like Fig. 5 finding, the keratinocyte of handling with CeLLGUARD does not demonstrate the obvious change of cell surface marker, and this discloses the basic characteristic that CeLLGUARD does not change cell.
Embodiment 6: the affirmation of the stem cell ability of keratinocyte
Handle from the isolating keratinocyte of skin histology 1 hour with control medium or CeLLGUARD, and get 500 cell inoculations back on 6 orifice plates and cultivated 7 days.In order to measure CFU, with taking out fully in the culture medium slave plate.The 1:1 mixture solution pair cell dyeing that adds Luo Danhong and Nile blue is after 30 minutes, to the colony counting number.
Fig. 6 has compared from the isolating keratinocyte of skin histology after handling with control medium or CeLLGUARD, the stem cell ability after the cultivation.Like Fig. 6 finding, the CFE comparison that CeLLGUARD obtains is high more than 4 times according to culture medium.This means that CeLLGUARD has produced the stem cell ability that significantly improves.
The theme that the present invention comprises is relevant with the korean patent application No.10-2010-0018122 that submitted in Korea S Department of Intellectual Property on February 26th, 2010, incorporates it into this paper at this through the entirety of quoting this application.
Although be to describe the present invention to the specific embodiment, it will be apparent to one skilled in the art that spirit and the scope that to carry out various changes and variation and not deviate from accompanying claims qualification of the present invention.
Industrial applicibility
As said, cytoprotective of the present invention can be used for the preservation cell safely, and not only can be used for storing stem cell but also can be used for storing various other zooblasts.This cytoprotective also can be used as and stores and transport the cell that is used for cell therapy at low temperatures.In addition, before cultivation,, can improve cell proliferation rate and stem cell ability through handling zooblast with cytoprotective of the present invention.Therefore, cytoprotective of the present invention is very useful as the preservation cell with the protective agent that improves the stem cell ability.
Claims (10)
1. a cytoprotective comprises aminoacid, mineral, vitamin, somatomedin and intacellin.
2. according to the cytoprotective of claim 1, wherein said aminoacid also comprises L-cystine and L-hydroxyproline except the basic aminoacid of constitutive protein matter.
3. according to the cytoprotective of claim 1, wherein said mineral also comprises trace element zirconium, germanium and vanadium except the required general element of cell growth.
4. according to the cytoprotective of claim 1, wherein said vitamin comprise can inhibitory reaction property oxygen species calcium pantothenate, choline chloride, folic acid, inositol and ascorbic acid.
5. according to the cytoprotective of claim 1, wherein said somatomedin comprises basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and platelet-derived growth factor (PDGF).
6. according to the cytoprotective of claim 1, wherein said intacellin is the extract of Placenta Hominis or the hydrolyzate of Placenta Hominis.
7. according to the cytoprotective of claim 1, said cytoprotective comprises the composition of listing among the table 1-4.
8. according to the cytoprotective of claim 1, said cytoprotective improves the viability at 0-37 ℃ of cell that stores down.
9. according to the cytoprotective of claim 1, said cytoprotective improves ability and the cell proliferation rate of stem cell but does not change cell characteristics.
10. according to the cytoprotective of claim 1, said cytoprotective be used to store with the transport cells therapeutic agent to strengthen the curative effect of said cellular therapeutic agent.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020100018122A KR101169701B1 (en) | 2010-02-26 | 2010-02-26 | Cell protectants comprising placenta extracts |
| KR10-2010-0018122 | 2010-02-26 | ||
| PCT/KR2010/002457 WO2011105658A1 (en) | 2010-02-26 | 2010-04-20 | Cell protectants comprising placenta extracts |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102791278A true CN102791278A (en) | 2012-11-21 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010800646536A Pending CN102791278A (en) | 2010-02-26 | 2010-04-20 | Cell protectants comprising placenta extracts |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101169701B1 (en) |
| CN (1) | CN102791278A (en) |
| WO (1) | WO2011105658A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| WO2018165997A1 (en) * | 2017-03-16 | 2018-09-20 | 杨涛 | Formula of serum-free medium for human pluripotent stem cells |
| CN108899106A (en) * | 2018-07-10 | 2018-11-27 | 长沙健金电子技术有限公司 | It is a kind of for protecting the devices and methods therefor of cell |
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| CN108424875A (en) * | 2018-04-20 | 2018-08-21 | 协和华东干细胞基因工程有限公司 | A kind of human archeocyte culture medium and preparation method thereof |
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| EP1181865A1 (en) * | 2000-08-23 | 2002-02-27 | Universite Catholique De Louvain | Cryoprotective solutions |
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| WO2008148938A1 (en) * | 2007-06-05 | 2008-12-11 | Kristiina Rajala | Formulations and methods for culturing embryonic stem cells |
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| KR100701297B1 (en) * | 2005-11-25 | 2007-03-29 | 신준호 | Culture Method of Fibroblast Using Placenta Extract and Composition for Skin Regeneration Using the Same |
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| EP1067138A1 (en) * | 1998-03-16 | 2001-01-10 | Japan Bioproducts Ind. Co., Ltd. | Hydroxyproline derivatives |
| CN1276152A (en) * | 1999-06-02 | 2000-12-13 | Mg制药株式会社 | Agent and method for preserving animal cells and organs |
| EP1181865A1 (en) * | 2000-08-23 | 2002-02-27 | Universite Catholique De Louvain | Cryoprotective solutions |
| WO2007061205A1 (en) * | 2005-11-25 | 2007-05-31 | Jun Ho Shin | Culture method of fibroblast using placenta extract and composition for skin regeneration using the same |
| WO2008148938A1 (en) * | 2007-06-05 | 2008-12-11 | Kristiina Rajala | Formulations and methods for culturing embryonic stem cells |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| CN106754714B (en) * | 2016-11-11 | 2020-05-19 | 北正赛欧（北京）生物科技有限公司 | Umbilical cord blood sample diluent, kit and method for processing umbilical cord blood to obtain stem cells |
| WO2018165997A1 (en) * | 2017-03-16 | 2018-09-20 | 杨涛 | Formula of serum-free medium for human pluripotent stem cells |
| US11485955B2 (en) | 2017-03-16 | 2022-11-01 | Tao Yang | Formula of serum-free medium for human pluripotent stem cells |
| CN108899106A (en) * | 2018-07-10 | 2018-11-27 | 长沙健金电子技术有限公司 | It is a kind of for protecting the devices and methods therefor of cell |
Also Published As
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| KR20110098478A (en) | 2011-09-01 |
| KR101169701B1 (en) | 2012-07-30 |
| WO2011105658A1 (en) | 2011-09-01 |
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