CN109266601A - The method for constructing clinical fat stem cell bank - Google Patents
The method for constructing clinical fat stem cell bank Download PDFInfo
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- CN109266601A CN109266601A CN201810763745.1A CN201810763745A CN109266601A CN 109266601 A CN109266601 A CN 109266601A CN 201810763745 A CN201810763745 A CN 201810763745A CN 109266601 A CN109266601 A CN 109266601A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
Abstract
The invention discloses the methods of building clinical fat stem cell bank, and this method comprises the following steps: step a, adipose tissue is handled, and acquisition is fatty to do thin frozen stock solution；Step b, SVF cell is obtained, adipose tissue is washed, digestion, separates acquisition SVF cell；Step c, fat stem cell culture inhales when cell confluency reaches 80% or so by SVF cell inoculation into complete medium and abandons culture medium, after washing, pancreatin is added, pancreatin effect is terminated after attached cell separation, followed by secondary culture obtains clinical fat stem cell library；The fat stem cell library of the method method efficient stable building of building clinical fat stem cell bank of the invention is highly-safe.
Description
Technical field
The invention belongs to human stem cells, in particular to fat stem cell.
Background technique
Fat stem cell (adipose-derived stem cells, ADSCs), ADSC pluripotent cell are in recent years from rouge
Isolated a kind of stem cell with multi-lineage potential in fat tissue.The repair function of main recovery organization cell, promotees
Into the regeneration of cell, while restoring young face, physical function is also fully improved, and is effectively improved inferior health, early ageing etc.
Disease is really effective against aging from inside to outside.
SVF (Stromal Vascular Fraction), Chinese name vascular stroma component are by passing through liposuction, disappearing
Change, centrifugation, cell mass that the various kinds of cell mixture isolated from adipose tissue is formed.It is rich comprising quantity in SVF
Rich mescenchymal stem cell (ASCs), high concentration thrombocyte plasma (PRP), growth factor, red blood cell, leucocyte, lymphocyte,
Monocyte etc..The study found that have a kind of synergistic effect between various cells in the SVF of fresh separated, between them mutually
Autocrine is influenced and passed through, increases the repair ability and wound healing ability of tissue, safety, validity are high.The source SVF
The clinic that fat stem cell is established studies less unstable quality with fat stem cell library at present.
Summary of the invention
In order to solve the deficiency of above-mentioned existing various methods, it is dry thin that the object of the present invention is to provide a kind of building clinical fats
The method in born of the same parents library, this method comprises the following steps:
Step a, adipose tissue is handled, and acquisition is fatty to do thin frozen stock solution；
Step b, SVF cell is obtained, adipose tissue is washed, digestion, separates acquisition SVF cell；
Step c, fat stem cell culture, by SVF cell inoculation into complete medium, when cell confluency reaches 80% left side
It is inhaled when right and abandons culture medium, after washing, pancreatin is added, pancreatin effect, followed by secondary culture are terminated after attached cell separation,
Obtain clinical fat stem cell library；
According to an aspect of the present invention, a kind of method constructing clinical fat stem cell bank, the cell after above-mentioned steps c are provided
It can also be stored.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, fat does thin frozen stock solution
Cryopreservation methods are as follows: the freezing storing box, frozen stock solution and cryopreservation tube for taking pre-cooling, add the frozen stock solution of appropriate volume to it is containing cell from
In heart pipe, 10 drops are gently slowly instilled along tube wall, are shaked gently centrifuge tube and are mixed cell, add remaining frozen stock solution, every jelly
It deposits pipe and 1.5ml frozen stock solution is added, screw pipe and be placed on the program cooling that isopropanol is added being pre-chilled in, merging -80 rapidly
DEG C refrigerator overnight, next day move into liquid nitrogen container.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, wherein frozen stock solution are as follows: DMSO
It is added dropwise to serum free medium and is configured to the cells frozen storing liquid of 10%DMSO concentration.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, step b obtains SVF cell
Method are as follows:
It prepares adipose tissue and digests agent solution: according to the amount of fatty sample, preparing the human fat tissue digestion of certain volume
Agent solution, sterilizing filter filtration sterilization, for use；
QC detection: QC takes a little waste liquid to keep sample from sample in cryopreservation tube and freezes, and separately takes a nutrient agar culture dish, adds few
Amount sample waste liquid does Sterility testing；
It washs adipose tissue: sample cleaning solution being added into fatty sample liquid storage bottle, repeat 3~4 times, until giving up for wash-off
Until liquid is clarified；
Digestion adipose tissue: by being dispensed into centrifuge tube for the fat equalization after washing, the people of equivalent is added in each pipe
Adipose tissue digestive ferment is placed in 37 DEG C of constant-temperature shaking incubators under aseptic condition and digests 30~60min.
Fractionation of fatty cell: being put into centrifuge for the fat digested, is centrifuged 10min, removal upper layer oil with 1100rmp
Rouge, fat and waste liquid fall remaining adipose tissue with 100 μm of strainer filtering with DMEM suspension cell.After drawing a little filtering
Cell suspension, counted with blood-counter system, filtered cell be dispensed into different centrifugations by freezing and being inoculated with requirement
Guan Zhong, with 1100rpm centrifugation 8 minutes.
According to an aspect of the present invention, a kind of method constructing clinical fat stem cell bank, above-mentioned adipose tissue digestion are provided
The ingredient of agent are as follows: clostridiopetidase A IV, pancreas enzyme -EDTA complex, alpha1-antitrypsin and dimethyl sulfoxide.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, wherein cell count formula
For value × 106 n=WBC × extension rate.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, above-mentioned steps c, fat are dry
Cell culture processes are as follows:
Configure the composition of complete medium are as follows: insulin, hydrocortisone, parathyroid hormone, estradiol, testosterone, blood
Platelet derivative growth factor-AB, epidermal growth factor, basic fibroblast growth factor, L-Glutamine, 2- sulfydryl second
Alcohol, LIF ELISA, L-AA, biotin, dexamethasone, insulin-like growth factor and human stem cell growth because
Son composition, and be dissolved in DMEM/F12 cell culture medium；
Cell inoculation culture: the cell for being inoculated with part is discarded supernatant, and stays a small amount of stoste, flicks suspension cell with finger；
Add appropriate complete medium suspension cell by inoculation bottle number again, be distributed into 3 bottles of culture bottles, and is added in each culture bottle again
Appropriate complete medium screws culture bottle cap, is placed in 37 DEG C, 5%CO2Incubator culture.Liposuction is marked on the culture bottle of inoculation
People's name, coding and date set 37 DEG C, 5%CO2, saturated humidity incubator culture；After culture 3 days, pipette, which is inhaled, abandons upper layer training
Base is supported, new complete medium is added；Culture is placed on inverted microscope observation for 6 days, if cell reaches 80% converging state, then
It takes pictures；It is such as not up to 80% converging state, then replaces culture medium, observes on one side, continue to cultivate on one side.Whether visual inspection has thin
Bacterium is mixed into, and checks whether culture medium is muddy, has seen whether microorganism motion artifacts with microscope, and result is recorded in accordingly
In record；
Cell dissociation is passed on, is frozen: when cell confluency reaches 80% or so, after observation of taking pictures, culture bottle being put into cleaning
Then workbench is inhaled with pipette and abandons culture medium；PBS is washed 2 times, and 2ml0.25% pancreatin is added, finds patch under inverted microscope
After parietal cell separation, 2ml complete medium is added and terminates pancreatin effect, blood counting chamber counts, and result is recorded in accordingly
In record；
The cell in 2 bottles of culture bottles is taken, into 6 bottles of culture bottles, the cell of 1 bottle of culture bottle to be taken to be frozen with 1:3 passage.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, above-mentioned storage method is as follows:
Frozen stock solution is prepared: being taken 50mlDMSO to be slowly dropped into the serum replacement (GIBCO) of 450ml, after mixing, is used 15ml
Centrifuge tube packing freezes in -20 DEG C of refrigerators；
It is diluted, is transferred in the program temperature reduction box containing isopropanol immediately, and fast using frozen stock solution according to the cell quantity frozen
Speed is transferred to -80 DEG C of refrigerator overnights, and then the cell frozen is transferred in liquid nitrogen container, and has registered name, numbered, freeze
The information such as date, freeze-stored cell algebra.
According to an aspect of the present invention, a kind of method for constructing clinical fat stem cell bank is provided, step can also include step
Rapid d, the step d are detection.
Detailed description of the invention
It Fig. 1, is one embodiment of the invention fat stem cell growth curve chart；
Fig. 2, scheme for the detection of one embodiment of the invention P2 fat subsitutes third day cell cycle；
Fig. 3, one embodiment of the invention streaming Indexs measure result figure；
Fig. 4, one embodiment of the invention test (× 4) result microphoto at rouge；
Fig. 5, one embodiment of the invention heterotopic Osteogenesis (× 4) result microphoto；
Fig. 6, one embodiment of the invention test (× 20) result microphoto at cartilage；
The influence diagram of hADSC prepared by Fig. 7, one embodiment of the invention to each dosage group mouse temperature；
Fig. 8, for the canonical plotting of one embodiment of the invention testosterone and IL-2；
Fig. 9, × 100 (female control group) microphotos are dyed for one embodiment of the invention liver organization HE；
Figure 10, × 100 (female control group) microphotos are dyed for one embodiment of the invention renal tissue HE；
Figure 11, × 100 (female control group) microphotos are dyed for one embodiment of the invention spleen tissue HE；
Figure 12, × 100 (female control group) microphotos are dyed for one embodiment of the invention ovary tissue HE；
Figure 13, × 100 (male control group) microphotos are dyed for the dirty tissue HE of one embodiment of the invention；
Figure 14, × 100 (male control group) microphotos are dyed for one embodiment of the invention renal tissue HE；
Figure 15, × 100 (male control group) microphotos are dyed for one embodiment of the invention spleen tissue HE；
Figure 16, × 100 (male control group) microphotos are dyed for one embodiment of the invention testis tissue HE；
Figure 17, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention liver organization HE；
Figure 18, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention renal tissue HE；
Figure 19, × 10 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention spleen tissue HE；
Figure 20, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention ovary tissue HE；
Figure 21, × 100 (the suitable dosage group of male) microphotos are dyed for one embodiment of the invention liver organization HE；
Figure 22, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention renal tissue HE；
Figure 23, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention spleen tissue HE；
Figure 24, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention testis tissue HE；
Figure 25, × 100 (female high dose group) microphotos are dyed for one embodiment of the invention liver organization HE；
Figure 26, × 100 (female high dose group) microphotos are dyed for one embodiment of the invention renal tissue HE；
Figure 27, × 100 (female high dose group) microphotos are dyed for one embodiment of the invention spleen tissue HE；
Figure 28, × 100 (female high dose group) microphotos are dyed for one embodiment of the invention ovary tissue HE；
Figure 29, × 100 (male high dose group) microphotos are dyed for one embodiment of the invention liver organization HE；
Figure 30, × 100 (male high dose group) microphotos are dyed for one embodiment of the invention renal tissue HE；
It Figure 31, is one embodiment of the invention spleen tissue (male high dose group) microphoto；
Figure 32, × 100 (male high dose group) microphotos are dyed for one embodiment of the invention testis tissue HE；
Figure 33, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention hepatonecrosis HE；
Figure 34, × 100 (the suitable dosage group of female) microphotos are dyed for one embodiment of the invention hepatonecrosis HE.
Specific embodiment
The present invention will be further described below with reference to the drawings.Construct the fat stem cell preparation and detection of clinical application
System
The foundation of 1 fat stem cell cell bank of embodiment
One, cultivating system
1, fatty transportational process and client's screening；
Indication screening
It is required that hospital must sign informed consent form, 3 parts of a formula with customer.Customer, each portion of medical institutions, another is with mark
Originally laboratory is delivered.
Information collection
The acquisition of customer's personal information: customer's personal information, passing treatment history, family are consulted in a manner of inquiring and fill in a form by hospital
Genetic history, and whether have the information such as the abnormal conditions for infecting medical history and hematopoiesis or immune system.
Physical examination information.Client's physical examination information should include following project: HIV-1/2 antibody (hiv antibody), HBsAg (second
Liver surface antigen), anti-HCV (antibody to hepatitis C), ALT (transaminase), syphilis helicoid antibody.Detection method executes respectively
The existing examination criteria of the Ministry of Public Health.
2, adipose tissue processing method；
Autologous fat stem cell preparation section starts preceding preparation
Environment: preparation personnel should be between cell manipulation and super-clean bench carries out ultraviolet disinfection in advance, and irradiation time is no less than 30
Minute.The purification air-conditioning blower opening time is no less than 30 minutes.Whether inspection environment monitoring report is in limited period.It checks clean
Whether the temperature and humidity of environment, pressure difference are in prescribed limit, and negative relative should be presented in toxic operation pressure difference.
Material: preparation instrument, reagent and consumptive material require to warehouse to get according to each process.And according to instruction to its name of an article,
Source, lot number, quality, quantity etc. are checked, and after signature confirmation, enter clean area as required.
Record form: relative recording table needed for preparing preparation section enters clean area by material requirement.
Personnel: enter clean area after registration.
Equipment: intact and cleaned, disinfection.Measurement instrument is within calibrating validity period.
Fat stem cell preparation
Take out freezing storing box, frozen stock solution and the cryopreservation tube for posting label of pre-cooling.Add the frozen stock solution of appropriate volume to containing
In the centrifuge tube of cell, 10 drops are gently slowly instilled along tube wall, are shaked gently centrifuge tube and are mixed cell, add remaining freeze
Liquid.1.5ml frozen stock solution is added in every cryopreservation tube.It screws pipe and is placed on the program cooling that isopropanol is added being pre-chilled in, rapidly
- 80 DEG C of refrigerator overnights are placed in, next day moves into liquid nitrogen container.The composition of frozen stock solution: DMSO is added dropwise to serum free medium, is configured to
The cells frozen storing liquid of 10%DMSO concentration.
3, SVF cell preparation method；
It prepares adipose tissue and digests agent solution: according to the amount of fatty sample, preparing the human fat tissue digestion of certain volume
Agent solution, 0.22 μm of sterilizing filter filtration sterilization, for use.The ingredient of adipose tissue digestive pharmaceutical are as follows: clostridiopetidase A IV, pancreatin-
EDTA complex, alpha1-antitrypsin and dimethyl sulfoxide.
QC detection: QC takes a little waste liquid to keep sample from sample in 2ml cryopreservation tube and freezes, and separately takes a nutrient agar culture dish,
A small amount of sample waste liquid is added to do Sterility testing.
It washs adipose tissue: sample cleaning solution being added into fatty sample liquid storage bottle, repeat 3~4 times, until giving up for wash-off
Until liquid is clarified.The composition of sample cleaning solution are as follows: the 500mlPBS solution that the gentamicin of 1000U is configured to.
Digestion adipose tissue: by being dispensed into 50ml centrifuge tube for the fat equalization after washing, equivalent is added in each pipe
Human fat tissue digestive ferment, be placed in 37 DEG C of constant-temperature shaking incubators under aseptic condition and digest 30~60min.
Fractionation of fatty cell: being put into centrifuge for the fat digested, is centrifuged 10min, removal upper layer oil with 1100rmp
Rouge, fat and waste liquid fall remaining adipose tissue with 100 μm of strainer filtering with DMEM suspension cell.After drawing a little filtering
Cell suspension, counted with blood-counter system, filtered cell be dispensed into different centrifugations by freezing and being inoculated with requirement
Guan Zhong, with 1100rpm centrifugation 8 minutes.Cell count formula: value × 10 n=WBC6× extension rate.
4, stem cell culture method；
The composition of complete medium are as follows: insulin, hydrocortisone, parathyroid hormone, estradiol, testosterone, blood platelet
It is derivative growth factor-AB, epidermal growth factor, basic fibroblast growth factor, L-Glutamine, 2 mercapto ethanol, white
Blood disease inhibiting factor, L-AA, biotin, dexamethasone, insulin-like growth factor and human stem cell growth group
At, and be dissolved in DMEM/F12 cell culture medium.
Cell inoculation culture: the cell for being inoculated with part is discarded supernatant, and stays a small amount of stoste, flicks suspension cell with finger.
Add appropriate complete medium suspension cell by inoculation bottle number again, is distributed into 3 T75 culture bottles, and again in each culture bottle
Appropriate complete medium is added, screws culture bottle cap, is placed in 37 DEG C, 5%CO2 incubator culture.It is marked on the culture bottle of inoculation
Liposuction people name, coding and date set 37 DEG C, 5%CO2, saturated humidity incubator culture；After culture 3 days, pipette, which is inhaled, to be abandoned
Layer culture medium, is added new complete medium；Culture is placed on inverted microscope observation for 6 days, converges shape as cell reaches 80%
State is then taken pictures.It is such as not up to 80% converging state, then replaces culture medium, observes on one side, continue to cultivate on one side.Visual inspection is
It is no to there is bacterium to be mixed into, check whether culture medium is muddy, has seen whether microorganism motion artifacts with microscope, and result is recorded
In respective record；
Cell dissociation is passed on, is frozen: when cell confluency reaches 80% or so, after observation of taking pictures, culture bottle being put into cleaning
Then workbench is inhaled with pipette and abandons culture medium.PBS is washed 2 times, and 2ml0.25% pancreatin is added, finds patch under inverted microscope
After parietal cell separation, 2ml complete medium is added and terminates pancreatin effect, blood counting chamber counts, and result is recorded in accordingly
In record.
The cell in 2 bottles of T75 culture bottles is taken, into 6 bottles of T175 culture bottles, to take the cell of 1 bottle of T75 culture bottle with 1:3 passage
It is frozen, the every amount of freezing should be not less than 2,000,000/.
It is recommended that: each secondary culture is that 2:1 ratio is cultivated and frozen, to obtain more stem cell.Cell passes
It is commissioned to train to support and should be not higher than for 6 generations.
5, cell storage method；
Frozen stock solution is prepared: being taken 50mlDMSO to be slowly dropped into the serum replacement (GIBCO) of 450ml, after mixing, is used 15ml
Centrifuge tube packing freezes in -20 DEG C of refrigerators.
It is diluted, is transferred in the program temperature reduction box containing isopropanol immediately, and fast using frozen stock solution according to the cell quantity frozen
Speed is transferred to -80 DEG C of refrigerator overnights, and then the cell frozen is transferred in liquid nitrogen container, and registered customer name, number,
Freeze the information such as date, freeze-stored cell algebra.
It is recommended that: in the fat stem cell freeze-stored cell quantity after cell dissociation, P0 generation, are no less than 2,000,000/ml, and P1 generation is many
In 2,000,000/ml, P2 generation and cell cryopreservation quantity suggestion later are frozen with 5,000,000 multiple, but highest is not to be exceeded
20000000.
2 detection architecture of embodiment
6, cell Quality Control detection method:
6.1, the foundation of amplification curve
6.1.1, MTT preparation of reagents: weighing 0.5 gram of MTT, be dissolved in the PBS of 100ml, with 0.22 μm of membrane filtration to remove
Go the bacterium in solution, 5mg/ml reagent, put 4 DEG C and be kept in dark place.During preparation and preservation, the most handy aluminium of container
Foil paper encases.
6.1.2, fat stem cell: taking amplification to enter the P2 fat subsitutes stem cell of plateau, and concentration of cell suspension is shown in Table one,
It is added dropwise in 2.5 piece of 96 orifice plate, 200 μ l are added dropwise in every hole, and 96 orifice plate rims holes are filled with sterile PBS, are placed in carbon dioxide training
It supports and is cultivated in case, culture to third day changes the liquid once.
Table 1, fat stem cell suspension concentration
| Grouping | Cell concentration/hole | It repeats |
| 1 | Blank control group | 6 |
| 2 | 1000 | 6 |
| 3 | 5000 | 6 |
| 4 | 8000 | 6 |
| 5 | 10000 | 6 |
6.1.3, MTT detection is carried out to stem cell every for 24 hours, detected 5 days altogether
6.1.4, MTT solution (5mg/ml) 20 μ l is added in every hole, continues to cultivate 4h.
6.1.5, culture is terminated, culture solution in hole is carefully sucked.
6.1.6,150ul dimethyl sulfoxide is added in every hole, shakes 10 minutes, dissolves crystal sufficiently.In enzyme linked immunological
The light absorption value in each hole, such as Fig. 1 are measured at detector OD490nm.
As seen from the figure, best inoculum density should be 5000/hole, i.e. 15000/cm2, dry thin in 2 days initial fat
Born of the same parents are in the laundering period of cell growth, enter logarithmic growth phase, the 4th day beginning cell decreased growth, into logarithm at 2-4 days
Growth period.
6.2, the cell cycle is detected
P2 fat subsitutes stem cell is taken, after culture 3 days, cell confluency degree is about 90%, 1 after being fixed with 70% ice ethyl alcohol ×
105(100 μ l), PBS are washed twice, and PI dye liquor (containing RNase) 300 μ l are added, mixes, is protected from light incubation 30 minutes, flow cytometer
Detection, P2 fat subsitutes third day cell cycle as shown in Figure 2
It is 2.14 ± 0.34%, the G2/M phase that the ratio of G0/G1 phase, which is the ratio of 96.78 ± 0.51%, S phase, as the result is shown
Ratio is 1.09 ± 0.18%.Illustrate cessation of cell division, it was demonstrated that can reach when cultivating 3 days using the cell of this system culture
To plateau.
6.3, cell flow cytometer detection
Detection Immunophenotyping marker has: negative indication CD34, CD45, CD14 and HLA-DR；Positive indication
CD105、CD49d、CD73、CD90。
The cell of harvest is suspended with PBS, is transferred in 15ml centrifuge tube after piping and druming uniformly, indicates cell coding, cell
Algebra, cell concentration and customer name.Every kind of marker takes 1 × 10520 μ l of antibody is added in cell (100 μ l), mixes, is protected from light
It is incubated for 30 minutes, PBS is washed twice, flow cytomery.,
The positive indication of fat stem cell detection is above 95% as the result is shown, and negative indication is below 5%.Such as 2 He of table
Shown in Fig. 3.
Table 2, streaming Indexs measure result
6.4, infectious diseases detects
Cell supernatant after taking a little centrifugation is used for Sterility testing into 15ml centrifuge tube；15ml centrifuge tube separately is taken, is taken
Above-mentioned obtained cell is used for the detection of infectious disease a little.Specific Testing index is as follows:
Table 3, the detection of stem cell infectivity and its standard
6.5, fat stem cell differentiation capability detects
It is tested at rouge:
It takes P3 fat subsitutes mescenchymal stem cell to be inoculated in 24 orifice plates, is inoculated with the every hole inoculation 10 in 3 holes5A, mesenchyma is dry
Cell culture medium culture 2 days is to cell fusion state.It is cleaned once with PBS, is changed to fat cell induced medium (DMEM+
+ 5% calf serum of 100U/ml antibiotic (FBS)+0.1mmol/L 3-isobutyl-1-methylxanthine (IBMX)+0.1mmol/
L Indomethacin+10mg/L insulin+1umol/L dexamethasone) induction 12 days, every 3 days full doses change liquid.It is removed after induction
Supernatant, PBS are cleaned 3 times, and 4% 4 DEG C of paraformaldehyde fixation is overnight；PBS is cleaned 2 times, every time 2 minutes；First by aqueous sealing agent
Liquid is heated in 60 DEG C of warm water；Preparing oil red O dye liquor, according to oil red O preparation of reagents stock solution, oil red O: dilution=
5:2, preparation total volume are 3.5ml dye liquor, are filtered with filter paper stand-by；Dye liquor is added dropwise to the cell orifice plate after cleaning through PBS
In, make its uniform fold in cell surface, dyes 15 minutes, wash off dye liquor extra in orifice plate with PBS later；It is contaminated with liquid is redyed
Color 3~5 minutes, then rinsed 2 times with PBS；Add the fixed cell surface of aqueous sealing agent, microscopy；As shown in Figure 4.
Heterotopic Osteogenesis:
It takes P3 fat subsitutes mescenchymal stem cell to be inoculated in 24 orifice plates, is inoculated with the every hole inoculation 10 in 3 holes5A, mesenchyma is dry
Cell culture medium culture 2 days is to cell fusion state.It is cleaned once with PBS, is changed to osteogenic (+10 μ g/ of DMEM in high glucose
L transforminggrowthfactor-β1+100nmol/L dexamethasone+6.25mg/L insulin+50nmol/L transferrins β 1+1% tire ox blood
Clearly), it induces 12 days, every 3 days full doses change liquid.Supernatant is removed after induction, PBS is cleaned 3 times, 4% 4 DEG C of paraformaldehyde fixation
Overnight；PBS is cleaned 2 times, every time 2 minutes；1% Alizarin red staining 30min washes away the extra dye liquor on creep plate with distilled water.Again
It is rinsed 2 times with PBS；Add the fixed cell surface of aqueous sealing agent, microscopy；As shown in Figure 5.
It is tested at cartilage:
It takes P3 fat subsitutes mescenchymal stem cell to be inoculated in 24 orifice plates, is inoculated with the every hole inoculation 10 in 3 holes5A, mesenchyma is dry
Cell culture medium culture 2 days is to cell fusion state.It is cleaned once with PBS, is changed into Cartilage culture base (DMEM+10% tire
+ 10 μ g/L of cow's serum+2mg/L insulin+3mg/L transferrins+1mmol/L pyruvic acid+10nmol/L dexamethasone conversion life
Long factor-beta) induction 12 days, every 3 days full doses change liquid.Supernatant is removed after induction, PBS is cleaned 3 times, 4% 4 DEG C of paraformaldehyde
It is fixed to stay overnight；PBS is cleaned 2 times, every time 2 minutes；0.1% toluidine blue liquid disseminates 20min.It is washed away with distilled water more on creep plate
Remaining dye liquor.It is rinsed 2 times with PBS again；Add the fixed cell surface of aqueous sealing agent, microscopy；As a result as shown in Figure 6.
Testing result shows that fat stem cell has the biological efficacy for being induced to differentiate into rouge, bone, cartilage, illustrates cell
Induce the versatility of differentiation.
6.6, cell paracrine detects
Each generation cell is taken, the content of flow cytomery secretory protein is used.Every hole adds 100 μ L of standard items or sample,
37 DEG C are incubated for 90 minutes；Liquid is discarded, 100 μ L of biotinylated antibody working solution is added in every hole, and 37 DEG C are incubated for 1 hour；Board-washing 3 times
Enzyme 100 μ L of conjugate working solution, 37 DEG C are incubated for 30 minutes.Board-washing 5 times, every hole adds 100 μ L of substrate solution, and 37 DEG C are protected from light incubation
15 minutes or so, add 50 μ L of terminate liquid, detects EGF, bFGF, 1 cytokine content of TGF-β (mean ± SD) with microplate reader
Table four, cytokine content (mean ± SD)
Testing result shows that fat stem cell can secrete the growth factors such as EGF, bFGF and TGF-β 1.And cell algebra pair
Sample content influences smaller.
6.7, animal safety detects
30 5~6 week old BALB/c-nu nude mices (weight 20g ±), half male and half female adapt to laboratory condition and raise one week.
Record animal heat, weight and forage volume before injection.
Table five, zoopery grouping and cell infusion dosage
Record animal heat, weight and forage volume before injection.
Cell suspension, trypan blue detect cell viability > 95%.Tail vein injection, fat stem cell are carried out by grouping dosage
Using finishing in 2 hours, control group gives respective volume physiological saline.
Clinical observation:
General status: situations such as observing animal skin, behavior, mouth and nose secretion, feed, drinking-water, excretion daily is surveyed daily
Measure and record weight, body temperature and food consumption variation and whether tumorigenesis.
Animal dead situation: carrying out pathological anatomy to all dead animals, and record symptom time of occurrence, duration,
It is death time, whether reversible etc..
Clinicopathologia assessment:
All animals are put to death for 14 days after stem cell injection, carry out clinicopathologia assessment.It extracts eyeball of mouse and takes blood, into
Row hematology and Serology biological chemical detection.
Hematology measurement:
Red blood cell (RBC) counts, leucocyte (WBC) counts, blood platelet (PLT) counts；Arneth's count, blood red egg
White (HB) measurement.
Plasma Biochemical detection:
Full automatic biochemical apparatus measures glutamic-pyruvic transaminase, urea nitrogen, creatinine, glucose, albumin, globulin and Archon than becoming
Change, ELISA method measures IL-2, testosterone levels variation.
Whole body main organs pathologic finding:
Observe internal organs shape, color, the quality such as brain, the heart, lung, liver,spleen,kidney, thyroid gland/parathyroid gland, testis/ovary etc.
Visually visible change, and weigh, calculate organ index.Liver,kidney,spleen and testis/ovary are taken, is fixed through formalin, stone
Wax slice, HE dyeing, carries out pathological observation.
Diagnostic criteria:
1, clinical manifestation diagnostic criteria:
1, situations such as hair, behavior, mouth and nose secretion, feed, drinking-water, excretion, should be normal；
2, body temperature, weight are without significant change；
2, clinicopathologia diagnostic criteria:
2.1 pathology evaluation standards:
A, blood routine is counted without significant change
B, Serum Indexes and serum cytokines are without significant change；
Histopathologic diagnostic criteria:
A, liver histopathology diagnostic criteria are as follows: lobuli hepatis is wide complete, liver cell marshalling, and fat-free denaturation is without extravasated blood
Deng；
B, renal pathology diagnostic criteria are as follows: normal in size, Renal vascular is without extravasated blood, and sinus renalis, calyces renales minores, glomerulus are without change
Shape occurs without idiozome, and weight is without significant difference.
C, spleen histopathological diagnosis standard are as follows: splenic capsule does not thicken, and spleen tissue is without extravasated blood, no lymph node hyperplasia.
D, genital system tissue pathological diagnosis standard are as follows: female organ changes without smooth muscle fibers, different developmental phases
Ovarian follicle, corpus luteum and lean type it is normal；Male organs Seminiferous Epithelium Structure is complete, and reproductive organs is without exception.
Experimental result
The variation of 1.1 mouse ordinary circumstances
It is observed continuously after tail vein injection hADSC 14 days, does not find the abnormal behaviour and death condition of Naked mouse.With compare
Group compares, and each test group of animals feed and drinking-water, body temperature and weight are all without significant changes (being shown in Table 6.2,6.3 and Fig. 7).It says
Bright hADSC tail vein injection has not significant impact the ordinary circumstances such as Naked mouse diet, body temperature and weight.
The influence (mean ± SD) that table 6.2.hADSC feeds nude mice and drinks water
Note: compared with same gender control group, P > 0.05.
Influence (mean ± SD) of the table 6.3:hADSC to nude mice body weight increase
Note: compared with same gender control group, P > 0.05.
1. pathology evaluation
The variation that 1.1 mouse haemocytes routinely count
After tail vein injection hADSC, after being observed continuously 14 days, plucks eyeball and take blood, carry out blood count conventional detection, with
Normal group compares, each dosage group female and male mice leukocyte differential count, red blood cell, hemoglobin and platelet count
Value without conspicuousness change (p>0.05), the white blood cell count(WBC) of only female mice high dose group significantly reduce (p<
0.05).Illustrate that hADSC routinely counts nude mice haemocyte does not influence substantially, high dose group female mice white blood cell count(WBC)
It reduces.
Table 4-1:hADSC injects the influence (mean ± SD) to nude mice blood count
Note: * is compared with identical gender control group, P < 0.05
Table 4-2:hADSC injects the influence (mean ± SD) to nude mice blood count
The variation of 1.2 mice serum indexs
The 14th day after tail vein injection hADSC, plucks eyeball and take blood, take serum after centrifugation, carried out using full automatic biochemical apparatus
Related serological Indexs measure.Table 5 the experimental results showed that, compared with female control group, fit dosage group glutamic-pyruvic transaminase ALT
It reduces, blood glucose GLU is increased, and high dose group urea UREA is increased.Compared with male control group, high dose group total protein TP, ball
Protein G LOB, urea UREA are reduced.Suitable dosage group urea UREA and creatinine CREA is reduced.But these amplitudes of variation are smaller,
Within normal range (NR).
Table 5-1:hADSC injects the influence (mean ± SD) to Naked mouse Biochemical Indices In Serum
Note: * is compared with identical gender control group, P < 0.05
Table 5-2:hADSC injects the influence (mean ± SD) to Naked mouse Biochemical Indices In Serum
Note: * is compared with identical gender control group, P < 0.05
The variation of 1.3 mice serum cell factors
It the 14th day after tail vein injection hADSC, plucks eyeball and takes blood, take serum after centrifugation, detect testis using ELISA kit
The variation of ketone and IL-2, standard curve are shown in Fig. 8.Compared with identical gender control group, the level of each dosage group testosterone and IL-2 do not have
There is significant change (see the table below 6).
Table 6:hADSC injects the influence (mean ± SD) to Naked mouse cell factor index
The variation of 1.4 mouse systemic main organs indexes
The 14th day after tail vein injection hADSC, mouse dissection is put to death, brain, the heart, lung, liver,spleen,kidney, first shape are visually observed
Internal organs shape, color, the quality such as gland/parathyroid gland, testis/ovary etc. do not have significant change, organ index the results are shown in Table 7-1,
Table 7-2 table 7-3.Compared with female control animals, heart, lungs, liver, kidney and the thymus gland of high dose group Female nude mice
Equal internal organs device index reduces (p < 0.05).But compared with male control group animal, the internal organs of each dosage group male Naked mouse refer to
The no significant change (p > 0.05) of number.
Table 7-1:hADSC injects the influence (mean ± SD) to Naked mouse organ index
Note: * is compared with identical gender control group, P < 0.05
Table 7-2:hADSC injects the influence (mean ± SD) to Naked mouse organ index
Note: * is compared with identical gender control group, P < 0.05
Table 7-3:hADSC injects the influence (mean ± SD) to Naked mouse organ index
Note: * is compared with identical gender control group, P < 0.05
2.5 mouse tissue pathology routine inspection results
The 14th day after tail vein injection hADSC, mouse dissection is put to death, the tissue taken is fixed on 10% formalin solution
In, according to routine paraffin wax microsection manufacture method, by materials, finishing, dehydration, embedding, slice, carry out conventional bush
Element-Yihong (Hematoxylin-Eosin, HE) dyeing.It is placed under optical microscopy and carries out the pathological study of sample.Group
Knit pathological observation result such as following figure Fig. 9 to Figure 34
It is described under tissue mirror:
Liver organization: the lobuli hepatis profile in each group is complete, hepatic cell cords marshalling, and liver cell has no that obvious fat becomes
Property, sinus hepaticus is slight extravasated blood, portal area blood vessel is slight extravasated blood.It was found that there is spotty necrosis, broken in 1 Female nude mice hepatic tissue of group in right amount
Sheet necrosis.Liver organization in control group, suitable dosage group and high dose group has no apparent metering the change of divergence.
Renal tissue: the Malpighian corpuscle size in each group is as usual, and a small amount of red blood cell of glomerulus has no glomus hyaloid
Become, have no that crescent occurs in Bowman's capsule, renal cells have no that vacuole becomes, and have no that various casts, interstitial blood vessel become silted up
Blood.Renal tissue in control group, suitable dosage group and high dose group has no apparent metering the change of divergence.
Spleen tissue: the splenic capsule in each group, which has no, to be thickened, red pulp is slight extravasated blood, white pulp relative decrease, white pulp endolymph
Brief summary has no obvious hyperplasia.Spleen tissue in control group, suitable dosage group and high dose group has no apparent metering the change of divergence.
Gonad: ovarian follicle, corpus luteum and the lean type of the visible different developmental phases of ovary in female each group, matrix therebetween are thin
Born of the same parents, reticular fibre and the smooth muscle fibers no abnormality seen being dispersed in change；The production of sperm of the visible seminiferous tubule of testis in male each group
Epithelial structure is complete, and sertoli cell and androgone no abnormality seen change, and visible sperm in lumen has no a large amount of abnormal morphologies
Sperm.Reproduction glandular tissue in control group, suitable dosage group and high dose group has no apparent metering the change of divergence.
Histopathological diagnosis:
The tissue of each dosage group has no that apparent abnormal pathologic changes, and the Pathologic changes of part Naked mouse may be individual
Factor causes, and does not measure the change of divergence significantly.
Three, experiment conclusions
14 days after tail vein injection hADSC, compared with the other control group of the same sex, the work of appropriate group and high dose group Naked mouse
Dynamic, body temperature, diet and weight etc. have no apparent anomalous variation；Routine hematology detection and the variation of biochemical indicator are also just
Within the scope of often；Testosterone and IL-2 are also without significant change.Compared with identical gender control group, high dose group Female nude mice
The organ indexs such as heart, lungs, liver, kidney and thymus gland reduce, and the organ index of male group does not have significant change, each dosage group
Tissue have no that apparent abnormal pathologic changes, the Pathologic changes of part Naked mouse may cause for individual factors, not show
The metering difference of work.Originally the experimental results showed that, hADSC tail vein injection does not cause the apparent acute toxicity performance of nude mice.
Following embodiments is some preferred embodiments in order to further illustrate the present invention, and not all embodiments.This
Field professional made under the premise of no progress creative work based on the other embodiment of the present invention, belong to this
The rights protection scope of invention.
Claims (10)
1. the method for constructing clinical fat stem cell bank, which is characterized in that described method includes following steps:
Step a, adipose tissue is handled, and acquisition is fatty to do thin frozen stock solution；
Step b, SVF cell is obtained, adipose tissue is washed, digestion, separates acquisition SVF cell；
Step c, fat stem cell culture, by SVF cell inoculation into complete medium, when cell confluency reaches 80% or so
It inhales and abandons culture medium, after washing, pancreatin is added, pancreatin effect, followed by secondary culture are terminated after attached cell separation, is obtained
Clinic uses fat stem cell library.
2. the method for building clinical fat stem cell bank according to claim 1, which is characterized in that thin after the step c
Born of the same parents can also store.
3. the method for building clinical fat stem cell bank according to claim 1, which is characterized in that the fat does thin
The cryopreservation methods of frozen stock solution are as follows: the freezing storing box, frozen stock solution and cryopreservation tube for taking pre-cooling add the frozen stock solution of appropriate volume thin to containing
In the centrifuge tube of born of the same parents, 10 drops are gently slowly instilled along tube wall, are shaked gently centrifuge tube and are mixed cell, add remaining freeze
1.5ml frozen stock solution is added in liquid, every cryopreservation tube, screws pipe and is placed on the program cooling that isopropanol is added being pre-chilled in, rapidly
- 80 DEG C of refrigerator overnights are placed in, next day moves into liquid nitrogen container.
4. the method for building clinical fat stem cell bank according to claim 3, which is characterized in that frozen stock solution are as follows: DMSO
It is added dropwise to serum free medium and is configured to the cells frozen storing liquid of 10%DMSO concentration.
5. the method for building clinical fat stem cell bank according to claim 1, which is characterized in that the step b is obtained
The method of SVF cell are as follows:
Prepare adipose tissue and digest agent solution: according to the amount of fatty sample, the human fat tissue digestive pharmaceutical for preparing certain volume is molten
Liquid, sterilizing filter filtration sterilization, for use；
QC detection: QC takes a little waste liquid to keep sample from sample in cryopreservation tube and freezes, and separately takes a nutrient agar culture dish, adds a small amount of mark
This waste liquid does Sterility testing；
It washs adipose tissue: sample cleaning solution being added into fatty sample liquid storage bottle, repeat 3~4 times, until the waste liquid of wash-off is clear
Until clear；
Digestion adipose tissue: by being dispensed into centrifuge tube for the fat equalization after washing, people's fat of equivalent is added in each pipe
Tissue digestion enzyme is placed in 37 DEG C of constant-temperature shaking incubators under aseptic condition and digests 30~60min.
Fractionation of fatty cell: being put into centrifuge for the fat digested, is centrifuged 10min with 1100rmp, removes upper layer grease, rouge
Fat and waste liquid fall remaining adipose tissue with 100 μm of strainer filtering with DMEM suspension cell.It draws a little filtered thin
Born of the same parents' suspension, is counted with blood-counter system, and filtered cell is dispensed into different centrifuge tubes by freezing and being inoculated with requirement,
With 1100rpm centrifugation 8 minutes.
6. the method for building clinical fat stem cell bank according to claim 5, it is characterised in that: the adipose tissue disappears
The ingredient of agent are as follows: clostridiopetidase A IV, pancreas enzyme -EDTA complex, alpha1-antitrypsin and dimethyl sulfoxide.
7. the method for building clinical fat stem cell bank according to claim 5, it is characterised in that: cell count formula is
Value × 106 n=WBC × extension rate.
8. the method for building clinical fat stem cell bank according to claim 1, it is characterised in that: step c, fat is dry thin
Born of the same parents' cultural method are as follows:
Configure the composition of complete medium are as follows: insulin, hydrocortisone, parathyroid hormone, estradiol, testosterone, blood platelet
It is derivative growth factor-AB, epidermal growth factor, basic fibroblast growth factor, L-Glutamine, 2 mercapto ethanol, white
Blood disease inhibiting factor, L-AA, biotin, dexamethasone, insulin-like growth factor and human stem cell growth group
At, and be dissolved in DMEM/F12 cell culture medium；
Cell inoculation culture: the cell for being inoculated with part is discarded supernatant, and stays a small amount of stoste, flicks suspension cell with finger；It presses again
Inoculation bottle number adds appropriate complete medium suspension cell, is distributed into 3 bottles of culture bottles, and is added in each culture bottle again appropriate
Complete medium screws culture bottle cap, is placed in 37 DEG C, 5%CO2Incubator culture.Liposuction people surname is marked on the culture bottle of inoculation
Name, coding and date, set 37 DEG C, 5%CO2, saturated humidity incubator culture；After culture 3 days, pipette, which is inhaled, abandons upper layer culture
New complete medium is added in base；Culture is placed on inverted microscope observation for 6 days, if cell reaches 80% converging state, then claps
According to；It is such as not up to 80% converging state, then replaces culture medium, observes on one side, continue to cultivate on one side.Whether visual inspection has bacterium
It is mixed into, checks whether culture medium is muddy, has seen whether microorganism motion artifacts with microscope, and result is recorded in corresponding note
In record；
Cell dissociation is passed on, is frozen: when cell confluency reaches 80% or so, after observation of taking pictures, culture bottle being put into clean work
Then platform is inhaled with pipette and abandons culture medium；PBS is washed 2 times, and 2ml0.25% pancreatin is added, and is found under inverted microscope adherent thin
After born of the same parents' separation, 2ml complete medium is added and terminates pancreatin effect, blood counting chamber counts, and result is recorded in respective record
In；
The cell in 2 bottles of culture bottles is taken, into 6 bottles of culture bottles, the cell of 1 bottle of culture bottle to be taken to be frozen with 1:3 passage.
9. the method for building clinical fat stem cell bank according to claim 2, it is characterised in that: the storage method is such as
Under:
Frozen stock solution prepare: take 50ml DMSO to be slowly dropped into the serum replacement (GIBCO) of 450ml, after mixing, with 15ml from
The packing of heart pipe freezes in -20 DEG C of refrigerators；
It is diluted, is transferred in the program temperature reduction box containing isopropanol immediately, and turn rapidly using frozen stock solution according to the cell quantity frozen
Enter to -80 DEG C of refrigerator overnights, then the cell frozen is transferred in liquid nitrogen container, and has registered name, numbered, freeze day
The information such as phase, freeze-stored cell algebra.
10. the method for building clinical fat stem cell bank according to claim 1, constructs the side of clinical fat stem cell bank
Method, which is characterized in that the step can also include step d, and the step d is detection.
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