CN104789522A - Construction method for human hair follicle stem cell bank - Google Patents
Construction method for human hair follicle stem cell bank Download PDFInfo
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- CN104789522A CN104789522A CN201510199001.8A CN201510199001A CN104789522A CN 104789522 A CN104789522 A CN 104789522A CN 201510199001 A CN201510199001 A CN 201510199001A CN 104789522 A CN104789522 A CN 104789522A
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Abstract
The invention discloses a construction method for a human hair follicle stem cell bank. The method comprises the steps of collecting the hair of a donor, acquiring and separating human hair follicle stem cells, cultivating the human hair follicle stem cells, detecting and cryopreserving the human hair follicle stem cells, constructing the bank and the like. According to the method, the human hair follicle stem cells can be effectively separated, purified and amplified, the current situation that such resources cannot be fully utilized is changed, and the stem cell bank for storing the human hair follicle stem cells is established to provide a large number of stem cell resources for clinical and scientific research application; the stem cell bank is constructed according to the acquired human hair follicle stem cells, and is used for storing hair follicle stem cells of an healthy adult and providing clinically applicable human hair follicle stem cells when the healthy adult requires stem cell treatment when being ill, and the stored human hair follicle stem cells can be used for producing a stem cell preparation with permission.
Description
Technical field
The present invention relates to the adult stem cell storehouse in a kind of human hair follicle source, i.e. the construction process in people's hair follicle stem cells storehouse.
Background technology
Stem cell (stem cell, SC) initiating cell with self, hyperproliferation and multi-lineage potential is referred to, embryonic stem cell (embryonic stem cells, ESCs) and adult stem cell (adult stem cells, ASCs) can be divided into according to differentiation capability.Embryonic stem cell and Almightiness type stem cell, most researching value on differentiation capability, but it derives from embryo, certainly will there is the problem of ethics aspect and security aspect (potential tumorigenicity), so embryonic stem cell is restricted in application.Though adult stem cell is weaker than embryonic stem cell on differentiation capability, but it avoids the problem of ethics aspect and security aspect, and autologous adult stem cells is transplanted, also immune rejection problems can be solved, avoid the injury that prolonged application immunosuppressor causes, so be better than embryonic stem cell from adult stem cell clinical application.
Adult stem cell is mainly present in can in the tissue of self and organ, skin histology upgrade and regenerative power by force, and hair follicle is as its accessory organ, remains the ability of cyclical growth and self all the life.Research shows, hair follicle is present in unique class embryonic tissue outside embryonic tissue so far, has the advantage of embryonic stem cell and adult stem cell concurrently.There is a large amount of pluripotent stem cell in hair follicle, can be smooth muscle cell, neuronal cell, neurogliocyte and melanocyte etc. through Differentiation Induction in vitro.So the research of hair follicle stem cells (hair follicle stem cells, HFSC) more and more receives the concern of people.
Hair follicle is a kind of attachment component of skin, is sunk formed by epidermis, is made up of epithelial hair root sheath and connective tissue sheath, is positioned at the hypomere of hair and surrounds hair root.With hair muscle on hair follicle, and be connected with sebiferous gland, sweat gland is also opened on hair follicle simultaneously.The origin of cell of epithelial hair root sheath is in epidermis, and connective tissue sheath originates from corium.Epithelial hair root sheath be recessedly go deep into corium by the epidermal area of skin, subcutaneous layer of fat forms, as epidermis extendible portion and hold hair, be divided into cuticle of root sheath, internal root sheath and external root sheath successively from inside to outside, external root sheath is equivalent to stratum basale and the stratum spinosum epidermidis of epidermis.In the external root sheath cell proliferation of hair shaft top, and form protuberance, become the mark of hair epimere.Cotsarelis etc. (1990) think that this bulge region around whole hair shaft top or can be only limitted to one-sided i.e. hair muscle attachment place, are considered to the main portions that hair follicle stem cells exists at present.
Bickenbach etc. (1981) have found label retaining cell (label retaining cell, LRC) in epiderm skin stratum basale, i.e. epidermal stem cells afterwards.After this, (2000) results of study such as Cotsarelis etc. (1990), Taylor find, also the existence of stem cell is had at the knuckle place being positioned at the hair follicle sidepiece under epidermis, they have larger proliferation potential and clonality, these stem cells can not only be transferred to hair follicle root downwards and generate hair follicle, and upwards can also move from raw coal bunker, generate epidermis and sebiferous gland.They are defined as hair follicle stem cells this cell, think that this multipotential stem cell may be the progenitor cell of epidermic cell, replace the research for the treatment of and gene therapy aspect significant to the cell of various dermatosis.
Stem cell in order to the mankind's different sources by acquisition is applied to following research and cell therapy, at present in world's country variant and area, the mankind have established multiple stem cell bank, such as umbilical cord mesenchymal stem cells, mesenchymal stem cells MSCs, navel blood stem cell etc.So-called stem cell bank is stored in the liquid nitrogen of-196 DEG C by stem cell and leaves the place of stem cell resource related data concentratedly in special frozen protection liquid.A perfect stem cell bank should possess preserves stem cell for a long time, maintains the fundamental characteristics of its stem cell, and the healthy stem cell stored can be provided the ability of Clinical practice whenever and wherever possible.Up to the present, other ripe adult stem cells develop into storehouse all, and due to people's hair follicle stem cells find more late, so there is no relevant people's hair follicle stem cells storehouse, therefore, success builds people's hair follicle stem cells storehouse, people's hair follicle stem cells of health is stored by the system engineering technology that the present invention sets up and builds storehouse, the effective ways that existing resource is made full use of, for storing me, family members and other people are when needs adopt people hair follicle stem cells to transplant and correlation technique treats various disease, clinical adaptive type people hair follicle stem cells is provided, there is very wide application prospect.
Summary of the invention
The object of the present invention is to provide a kind of construction process of people's hair follicle stem cells storehouse, aforesaid method have raw material sources extensively, obtain convenient, cost is low, have a extensive future, and can be the advantage that clinical and research provides a large amount of stem cell resource.
The construction process in a kind of people's hair follicle stem cells storehouse provided by the invention, comprises the following steps:
1) collection of donor hair: before gathering donor hair, carry out gathering the information at least comprising donor identity identification information; Then ABO/Rh Blood grouping, the detection of HLA somatotype and the detection of HIV, HBV, HCV are comprised to donor, then aseptically gather the hair of donor;
2) obtain and be separated people's hair follicle stem cells: preferably, aseptically, the hair that picking hair papilla is complete, clip hair follicle root, repeatedly rinsing 3-5 time with containing dual anti-equilibrated phosphate buffered soln; Then get hair follicle with aseptic ophthalmic tweezers sub-folder and put into 6 orifice plates, add hair follicle stem cells nutrient solution 600 μ L/ hole, 37 DEG C, 5%CO
2incubator in cultivate, fluid infusion next day to 3mL/ hole continue cultivate, within 2-3 days, change fresh hair follicle stem cells nutrient solution, every day, observation of cell growing state, obtained primary people's hair follicle stem cells in time growing to density and be 80-90%;
3) cultivation of people's hair follicle stem cells: preferably, by primary people's hair follicle stem cells of acquisition with 0.1 × 10
5-1.0 × 10
5individual cell/cm
2density be seeded in Tissue Culture Dish, add fresh hair follicle stem cells nutrient solution, be placed in 37 DEG C, 5%CO
2condition under cultivate, within every 2-3 days, change once new nutrient solution, until cell grow to density be 80-90% merge time, nutrient solution before removal, remove after adding aseptic equilibrated phosphate buffered soln cleaning, then the pancreatin of 0.25%-0.5% containing EDTA is added, 3-5 minute is digested under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion; Then postdigestive for termination people's hair follicle stem cells suspension is transferred in centrifuge tube, 800-1000 rev/min of centrifugal 5-6 minute, terminates rear removal supernatant liquor, and it is resuspended to add fresh hair follicle stem cells nutrient solution, last 1:3 or 1:4 goes down to posterity, and obtains more people's hair follicle stem cells;
4) detection of people's hair follicle stem cells and frozen:
The detection of people's hair follicle stem cells: preferably, until people's hair follicle cell grow to density be 80-90% merge time, nutrient solution before removal, remove after adding aseptic equilibrated phosphate buffered soln cleaning, then the pancreatin of 0.25%-0.5% containing EDTA is added, 3-5 minute is digested under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion; Then be transferred in centrifuge tube by postdigestive for termination people's hair follicle stem cells suspension, 800-1000 rev/min centrifugal 5 minutes, terminates rear removal supernatant; The some people hair follicle stem cells of results is carried out quality examination, and described quality examination comprises Sterility testing, detection of mycoplasma, cytoactive detection, differentiation capability detection and FCM analysis, to determine whether cell reaches criterion of acceptability; All the other carry out cell cryopreservation with batch people's hair follicle stem cells;
People's hair follicle stem cells frozen: preferably, utilize people's hair follicle stem cells frozen storing liquid resuspended people's hair follicle stem cells of same batch of above-mentioned acquisition, adjustment cell density is 1 × 10
6~ 1 × 10
7individual/mL, is distributed in cryopreservation tube, capping, and carries out numbering, carries out program mode cooling immediately, puts into the medium-term and long-term preservation of liquid nitrogen of-196 DEG C afterwards; Carry out all frozen records during cell cryopreservation, meet the standard of stem cell freezen protective, storage condition periodic detection;
Wherein, people's hair follicle stem cells detection and frozenly to carry out, namely after detected result out simultaneously, cell is frozen, at this moment will detect underproof cell according to its numbering, corresponding freeze-stored cell together to be discarded together with cryopreservation tube, and make a record, have technician to process afterwards;
5) storehouse is built: on the container carrier storing warehouse-in, set up relevant information, by the essential information of aforementioned gathered donor and the preparation of corresponding human hair follicle stem cells and the relevant information input computer database of cell bank building process thereof, and recall associated note by retrieve encoded and donor information examines, include the library management of people's hair follicle stem cells in, namely complete the structure in people's hair follicle stem cells storehouse.
Wherein, Tissue Culture Dish can also substitute with other same cells culture apparatus, and cryopreservation tube can also replace with the similar container of other non-tubing.Above-mentioned concrete volumetric quantities, the numerical value such as treatment time, number of times is only citing, instead of well-determined.
Wherein, described hair follicle stem cells nutrient solution be containing support the sugar of stem cell growth, amino acid, somatomedin, inorganic salt, trace element the DMEM/F12 nutrient solution of commerical prod, containing 10% foetal calf serum, bFGF 10ng/mL, dual anti-(100u/mL).
The aqueous solution of described equilibrated phosphate buffered soln mainly containing inorganic salt, can be PBS salt buffer solution, D-Hanks salt buffer solution, or SPB salt buffer solution etc., it can maintain Premeabilisation of cells pressure balanced, keeps pH to stablize.
Described people's hair follicle stem cells frozen storing liquid is for containing cell freezing protective material; general conventional DMSO class perviousness protective material; can damage from solute damage and ice crystal by Cell protection; simultaneously also containing serum and stem cell minimum medium; comprise DMEM, α-MEM etc.; wherein cryoprotectant (as DMSO), the proportioning of serum and stem cell minimum medium is 1:2:7.
Described program mode cool-down method comprises 4 DEG C and preserves 40-60min, and preserve 1-2h for-20 DEG C ,-80 DEG C are spent the night, and are finally deposited into-196 DEG C of liquid nitrogen, also can directly be placed in Programmed cryopreservation box, put into-80 DEG C of refrigerator overnight, within second day, forward in the liquid nitrogen of-196 DEG C.
Compared with prior art, beneficial effect of the present invention is as follows:
The first, utilize the inventive method to set up the stem cell bank storing people's hair follicle stem cells, this stem cell bank can provide a large amount of stem cell resource to clinical and research application;
The second, the stem cell bank that the people's hair follicle stem cells utilizing the inventive method to obtain is built into, its abundance, it is convenient that cell obtains, a large amount of and activated people's hair follicle stem cells can be obtained, and can preserve for a long time and not lose activity, have a good application prospect; This storehouse is healthy grownup's hair follicle stem cells, for storage person need under special circumstances adopt stem cell transplantation and correlation technique treatment time, people's hair follicle stem cells of clinical adaptive type is provided, and after obtaining permission, stored stem cell is used for the production of other various stem cell medicines.
Certainly, implement arbitrary product of the present invention might not need to reach above-described all advantages simultaneously.
Accompanying drawing explanation
Fig. 1 is step 2 in the embodiment of the present invention 1) the 100X inverted microscope figure of primary people's hair follicle stem cells of gained;
Fig. 2 be in the embodiment of the present invention 1 step 3) go down to posterity after the 100X inverted microscope figure of people's hair follicle stem cells of gained;
Fig. 3 is the FCM analysis figure of people's hair follicle stem cells in step 4) in the embodiment of the present invention 1, in figure, and A:CD105-APC; B:CD13-FITC; C:CD14-FTIC; G:CD34-PE; H:HLA-DR; I:CD90-PE;
Fig. 4 is the step 2 of the embodiment of the present invention 2) in the 100X inverted microscope figure of the primary people's hair follicle stem cells of gained;
The 100X inverted microscope figure of people's hair follicle stem cells of gained after going down to posterity in the step 3) of Fig. 5 embodiment of the present invention 2;
Fig. 6 is the FCM analysis figure of people's hair follicle stem cells in the step 4) of the embodiment of the present invention 2, in figure: A:CD105-APC; B:CD13-FITC; C:CD14-FTIC; G:CD34-PE; H:HLA-DR; I:CD90-PE.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be appreciated that, these embodiments only for illustration of the present invention, and are not intended to limit the scope of the invention.The improvement made according to the present invention of those skilled in the art and adjustment, still belong to protection scope of the present invention in actual applications.
Embodiment 1
Operation in the present embodiment is all carried out at rigorous aseptic environment.
The construction process in people's hair follicle stem cells storehouse that the present embodiment provides comprises the following steps:
1) collection of donor hair: before gathering donor hair, informed consent postscript need be signed through donor, can gather.Informed Consent Form comprises the information such as donor name, age, identification card number, address, contact method.After signing letter of consent, ABO/Rh Blood grouping, the detection of HLA somatotype and the detection of HIV, HBV, HCV are carried out to donor, then aseptically gathers the hair of donor; In other embodiments of the present invention, also can carry out except above-mentioned information acquisition and detect except picker think need gather information and needs other detect;
2) obtain and be separated people's hair follicle stem cells: aseptically, the hair that picking hair papilla is complete, clip hair follicle root, repeatedly rinsing 3 times with containing dual anti-PBS.Then get hair follicle with aseptic ophthalmic tweezers sub-folder and put into 6 orifice plates, add hair follicle stem cells nutrient solution (containing DMEM/F12,10%FBS, bFGF 10ng/mL, dual anti-100u/mL) 600 μ L/ hole, 37 DEG C, 5%CO
2incubator in cultivate, fluid infusion next day continues to 3mL/ hole to cultivate, within every 2 days, change fresh hair follicle stem cells nutrient solution, every day observation of cell growing state, primary people's hair follicle stem cells is obtained in time growing to 80-90%, the 100X inverted microscope figure of the primary people's hair follicle stem cells of gained as shown in Figure 1, can observe from this Fig. 1, and people's hair follicle stem cells that in the present embodiment, success is separated grows fine; Wherein, DMEM/F12 substratum is suitable for the cultivation of Clonal density.F12 medium component is complicated, containing various trace elements, and DMEM combines with 1:1, is called DMEM/F12 substratum (DME/F12medium), as the basis of exploitation serum-free formula, contain more rich composition to utilize F12 and DMEM contains the nutritive ingredient of higher concentration for advantage.This substratum is applicable to serum content comparatively mammaliancellculture under low condition.In order to strengthen the surge capability of this substratum, improvement one of be at DMEM/F12(1:1) in add 15mMHEPES damping fluid.
3) cultivation of people's hair follicle stem cells: by step 2) in obtain primary people's hair follicle stem cells with 0.1 × 10
5individual cell/cm
2density be seeded in Tissue Culture Dish, add fresh hair follicle stem cells nutrient solution, be placed in 37 DEG C, 5%CO
2condition under cultivate, the hair follicle stem cells nutrient solution more renewed for every 2 days, until cell grow to 80-90% merge time, nutrient solution before removal, removes after adding aseptic PBS cleaning, then adds the pancreatin that 0.25% contains EDTA, digest 3 minutes under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion.Postdigestive for termination people's hair follicle stem cells suspension is transferred in centrifuge tube, 1000 revs/min centrifugal 5 minutes, supernatant is removed after end, add fresh hair follicle stem cells nutrient solution resuspended, last 1:3 goes down to posterity, and obtains more people's hair follicle stem cells, and the 100X inverted microscope figure of the people's hair follicle stem cells obtained after going down to posterity as shown in Figure 2, can observe from this Fig. 2, people's hair follicle stem cells cultivation conditions of the present embodiment is good;
4) detection of people's hair follicle stem cells and frozen: wherein,
The detection method of people's hair follicle stem cells is as follows: the people's hair follicle stem cells after going down to posterity grow to density be 80-90% merge time, nutrient solution before removal, removes after adding aseptic PBS cleaning, then adds the pancreatin that 0.25% contains EDTA, digest 3 minutes under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion.Be transferred in centrifuge tube by postdigestive for termination people's hair follicle stem cells suspension, 1000 revs/min centrifugal 5 minutes, terminates rear removal supernatant.The some people hair follicle stem cells of results is carried out quality examination, comprise Sterility testing, detection of mycoplasma, cytoactive detection, differentiation capability detection and FCM analysis etc., to determine whether cell reaches criterion of acceptability, wherein the result of FCM analysis is carried out as shown in Figure 3 to the present embodiment gained cell, Fig. 3 can observe, the streaming surface antibody result detected meets the primary standard of mescenchymal stem cell, and all the other the same batch people's hair follicle stem cells not carrying out quality examination are carried out cell cryopreservation.
The cryopreservation methods of people's hair follicle stem cells is as follows; Utilize people's hair follicle stem cells frozen storing liquid resuspended people's hair follicle stem cells of same batch of above-mentioned acquisition, adjustment cell density is 1 × 10
6individual/mL, is distributed in cryopreservation tube, capping, and carries out numbering, carries out program mode cooling immediately, puts into the medium-term and long-term preservation of liquid nitrogen of-196 DEG C afterwards.Carry out all frozen records during cell cryopreservation, meet the standard of stem cell freezen protective, storage condition periodic detection.Wherein, the people's hair follicle stem cells frozen storing liquid used contains cell freezing protective material; general conventional this type of perviousness protective material of DMSO; can damage from solute damage and ice crystal by Cell protection; simultaneously also containing serum and stem cell minimum medium; comprise DMEM, α-MEM etc., wherein cryoprotectant (as DMSO), the proportioning of serum and stem cell minimum medium is 1:2:7.Wherein, above-mentioned program mode cool-down method can be such as: preserve 40-60min for 4 DEG C, and preserve 1-2h for-20 DEG C ,-80 DEG C are spent the night, and are finally deposited into-196 DEG C of liquid nitrogen; Or also can be, directly be placed in Programmed cryopreservation box, put into-80 DEG C of refrigerator overnight, within second day, forward in the liquid nitrogen of-196 DEG C.
Wherein, people's hair follicle stem cells detection and frozenly to carry out, namely after detected result out simultaneously, cell is frozen, at this moment will detect underproof cell according to its numbering, corresponding freeze-stored cell together to be discarded together with cryopreservation tube, and make a record, have technician to process afterwards.
5) storehouse is built: on the container carrier storing warehouse-in, set up relevant information, by the essential information of aforementioned gathered donor and the preparation of corresponding human hair follicle stem cells and the relevant information input computer database of cell bank building process thereof, and recall associated note by retrieve encoded and donor information examines, include the library management of people's hair follicle stem cells in, namely complete the structure in people's hair follicle stem cells storehouse.
Embodiment 2
The construction process in people's hair follicle stem cells storehouse that the present embodiment provides comprises the steps:
1) collection of donor hair: before gathering donor hair, informed consent postscript need be signed through donor, can gather.Informed Consent Form comprises the information such as donor name, age, identification card number, address, contact method.After signing letter of consent, ABO/Rh Blood grouping, the detection of HLA somatotype and the detection of HIV, HBV, HCV are carried out to donor, then aseptically gathers the hair of donor; In other embodiments of the present invention, also can carry out except above-mentioned information acquisition and detect except picker think need gather information and needs other detect;
2) obtain and be separated people's hair follicle stem cells: aseptically, the hair that picking hair papilla is complete, clip hair follicle root, repeatedly rinsing 5 times with containing dual anti-PBS.Then get hair follicle with aseptic ophthalmic tweezers sub-folder and put into 6 orifice plates, add hair follicle stem cells nutrient solution (DMEM/F12,10%FBS, bFGF 10ng/mL, dual anti-100u/mL) 600 μ L/ hole, 37 DEG C, 5%CO
2incubator in cultivate, fluid infusion next day continues to 3mL/ hole to cultivate, within every 3 days, change fresh hair follicle stem cells nutrient solution, every day observation of cell growing state, primary people's hair follicle stem cells is obtained in time growing to density and be 80-90%, the 100X inverted microscope figure of the primary people's hair follicle stem cells of gained as shown in Figure 4, can observe out from this Fig. 4, and people's hair follicle stem cells that the present embodiment is successfully separated grows fine; Wherein, DMEM/F12 substratum is suitable for the cultivation of Clonal density.F12 medium component is complicated, containing various trace elements, and DMEM combines with 1:1, is called DMEM/F12 substratum (DME/F12medium), as the basis of exploitation serum-free formula, contain more rich composition to utilize F12 and DMEM contains the nutritive ingredient of higher concentration for advantage.This substratum is applicable to serum content comparatively mammaliancellculture under low condition.In order to strengthen the surge capability of this substratum, improvement one of be at DMEM/F12(1:1) in add 15mMHEPES damping fluid.
3) cultivation of people's hair follicle stem cells: by step 2) primary people's hair follicle stem cells of obtaining is with 1.0 × 10
5individual cell/cm
2density be seeded in Tissue Culture Dish, add fresh hair follicle stem cells nutrient solution, be placed in 37 DEG C, 5%CO
2condition under cultivate, every nutrient solution more renewed for 3 days, until cell grow to density be 80-90% merge time, nutrient solution before removal, removes after adding aseptic PBS cleaning, then adds the pancreatin that 0.25% contains EDTA, digest 5 minutes under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion.Postdigestive for termination people's hair follicle stem cells suspension is transferred in centrifuge tube, 1000 revs/min centrifugal 5 minutes, supernatant is removed after end, add fresh hair follicle stem cells nutrient solution resuspended, last 1:4 goes down to posterity, and obtains more people's hair follicle stem cells, and the 100X inverted microscope figure of the people's hair follicle stem cells obtained after going down to posterity as shown in Figure 5, can observe out from this Fig. 5, people's hair follicle stem cells cultivation conditions of the present embodiment is good;
4) detection of people's hair follicle stem cells and frozen:
The detection of people's hair follicle stem cells: the people's hair follicle cell after going down to posterity grow to density be 80-90% merge time, remove old nutrient solution, remove after adding aseptic PBS cleaning, then add the pancreatin that 0.25% contains EDTA, digest 5 minutes under 37 DEG C of conditions, then the digestion situation of cell is examined under a microscope, with the beating culture dish that have gentle hands is light, to help cell detachment, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion.Be transferred in centrifuge tube by postdigestive for termination people's hair follicle stem cells suspension, 1000 revs/min centrifugal 5 minutes, terminates rear removal supernatant.The some people hair follicle stem cells of results is carried out quality examination, comprise Sterility testing, detection of mycoplasma, cytoactive detection, differentiation capability detection and FCM analysis etc., to determine whether cell reaches criterion of acceptability, wherein the result of the FCM analysis of the present embodiment gained cell as shown in Figure 6, can observe in Fig. 6, the streaming surface antibody result detected meets the primary standard of mescenchymal stem cell, and all the other the same batch people's hair follicle stem cells not carrying out quality examination are carried out cell cryopreservation;
People's hair follicle stem cells frozen: utilize people's hair follicle stem cells frozen storing liquid resuspended people's hair follicle stem cells of same batch of above-mentioned acquisition, adjustment cell density is 1 × 10
7individual/mL, is distributed in cryopreservation tube, capping, and carries out numbering, carries out program mode cooling immediately, puts into the medium-term and long-term preservation of liquid nitrogen of-196 DEG C afterwards.Carry out all frozen records during cell cryopreservation, meet the standard of stem cell freezen protective, storage condition periodic detection.Wherein, above-mentioned people's hair follicle stem cells frozen storing liquid contains cell freezing protective material; general conventional this type of perviousness protective material of DMSO; can damage from solute damage and ice crystal by Cell protection; simultaneously also containing serum and stem cell minimum medium; comprise DMEM, α-MEM etc., wherein cryoprotectant (as DMSO), the proportioning of serum and stem cell minimum medium is 1:2:7.In addition, above-mentioned program mode cool-down method can be such as: preserve 40-60min for 4 DEG C, and preserve 1-2h for-20 DEG C ,-80 DEG C are spent the night, and are finally deposited into-196 DEG C of liquid nitrogen; Or also can be, directly be placed in Programmed cryopreservation box, put into-80 DEG C of refrigerator overnight, within second day, forward in the liquid nitrogen of-196 DEG C.
Wherein, people's hair follicle stem cells detection and frozenly to carry out, namely after detected result out simultaneously, cell is frozen, at this moment will detect underproof cell according to its numbering, corresponding freeze-stored cell together to be discarded together with cryopreservation tube, and make a record, have technician to process afterwards;
5) storehouse is built: on the container carrier storing warehouse-in, set up relevant information, by the essential information of aforementioned gathered donor and the preparation of corresponding human hair follicle stem cells and the relevant information input computer database of cell bank building process thereof, and recall associated note by retrieve encoded and donor information examines, include the library management of people's hair follicle stem cells in, namely complete the structure in people's hair follicle stem cells storehouse.
The present invention has the following advantages:
The first, the stem cell bank of the storage people hair follicle stem cells utilizing the inventive method to set up can provide a large amount of stem cell resource to clinical and research application;
The second, utilize the stem cell bank that the inventive method builds, its abundance, it is convenient that cell obtains, and can obtain a large amount of and activated people's hair follicle stem cells, and can preserve for a long time and not lose activity, have a good application prospect; What this stem cell bank stored is the hair follicle stem cells of normal adults, for storage person need under special circumstances adopt stem cell transplantation and correlation technique treatment time, people's hair follicle stem cells of clinical adaptive type is provided, and after obtaining permission, stored stem cell is used for the production of other various stem cell medicines;
3rd. method of the present invention, can effectively be separated, purification and amplification people hair follicle stem cells, solve the present situation that this type of resource is not fully utilized, and set up out the stem cell bank of standing storage people hair follicle stem cells to provide a large amount of stem cell resources to clinical and research application.Stem cell bank is built according to the people's hair follicle stem cells obtained, this storehouse is that health adult stores hair follicle stem cells, for he or she ill need adopt stem-cell therapy time, clinical adaptive type people hair follicle stem cells is provided, and after obtaining license, stored people's hair follicle stem cells can be used for stem cell medicine and produce.
Under the instruction of the present invention and above-described embodiment, those skilled in the art are easy to predict, cited or each raw material that exemplifies of the present invention or its equivalent alterations, each working method or its equivalent alterations can realize the present invention, and the parameter bound value of each raw material and working method, interval value can realize the present invention, do not enumerate embodiment at this.
Claims (6)
1. the construction process in people's hair follicle stem cells storehouse, is characterized in that, comprises the following steps:
1) collection of donor hair: before gathering donor hair, carry out gathering the information at least comprising donor identity identification information; Then ABO/Rh Blood grouping, the detection of HLA somatotype and the detection of HIV, HBV, HCV are comprised to donor, then aseptically gather the hair of donor;
2) obtaining and be separated people's hair follicle stem cells: aseptically, the hair that picking hair papilla is complete, obtaining hair follicle root, obtaining hair follicle 3-5 time with repeatedly rinsing containing dual anti-equilibrated phosphate buffered soln; Then under aseptic conditions hair follicle is put into 6 orifice plates, add hair follicle stem cells nutrient solution 600 μ L/ hole, at 37 DEG C, 5%CO
2incubator in cultivate, fluid infusion next day to 3mL/ hole continue cultivate, every 2-3 days changes fresh hair follicle stem cells nutrient solution, in time growing to density and be 80-90%, obtain primary people's hair follicle stem cells;
3) cultivation of people's hair follicle stem cells: by step 2) primary people's hair follicle stem cells of obtaining is with 0.1 × 10
5-1.0 × 10
5individual cell/cm
2density be seeded in cell culture apparatus, add fresh hair follicle stem cells nutrient solution, be placed in 37 DEG C, 5%CO
2condition under cultivate, every 2-3 days changes fresh hair follicle stem cells nutrient solution, until cell grow to density be 80-90% merge time, the nutrient solution before removal, cleans with aseptic equilibrated phosphate buffered soln, then the pancreatin of 0.25%-0.5% containing EDTA is added, under 37 DEG C of conditions, digest 3-5 minute, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion; Then postdigestive for termination people's hair follicle stem cells suspension is transferred in centrifuge tube, 800-1000 rev/min of centrifugal 5-6 minute, terminates rear removal supernatant liquor, and it is resuspended to add fresh hair follicle stem cells nutrient solution, 1:3 or 1:4 goes down to posterity afterwards, obtains more people's hair follicle stem cells;
4) detection of people's hair follicle stem cells and frozen:
The detection of people's hair follicle stem cells: until people's hair follicle cell grow to density be 80-90% merge time, nutrient solution before removal, clean with aseptic equilibrated phosphate buffered soln, then the pancreatin of 0.25%-0.5% containing EDTA is added, under 37 DEG C of conditions, digest 3-5 minute, after cell all comes off, add isopyknic hair follicle stem cells nutrient solution, piping and druming is even, to stop digestion; Then postdigestive for termination people's hair follicle stem cells suspension is transferred in centrifuge tube, 800-1000 rev/min of centrifugal 5-6 minute, terminates rear removal supernatant; Carried out determining whether cell reaches the quality examination of criterion of acceptability by the some people hair follicle stem cells of results, described quality examination at least comprises Sterility testing, detection of mycoplasma, cytoactive detection, differentiation capability detection and FCM analysis; All the other carry out cell cryopreservation with batch people's hair follicle stem cells;
People's hair follicle stem cells frozen: utilize people's hair follicle stem cells frozen storing liquid resuspended people's hair follicle stem cells of same batch of above-mentioned acquisition, adjustment cell density is 1 × 10
6~ 1 × 10
7individual/mL, is distributed in cryopreservation tube, capping, and carries out numbering, carries out program mode cooling immediately, puts into the medium-term and long-term preservation of liquid nitrogen of-196 DEG C afterwards; Carry out all frozen records during cell cryopreservation, meet the standard of stem cell freezen protective, storage condition periodic detection;
Wherein, people's hair follicle stem cells detection and frozenly to carry out, namely after detected result out simultaneously, cell is frozen, at this moment will detect underproof cell according to its numbering, corresponding freeze-stored cell together to be discarded together with cryopreservation tube, and make a record, processed by technician afterwards;
5) storehouse is built: the relevant information setting up people's hair follicle stem cells on the container carrier storing warehouse-in, by the essential information of aforementioned gathered donor and the preparation of corresponding human hair follicle stem cells and the relevant information input computer database of cell bank building process thereof, and recall associated note by retrieve encoded and donor information examines, include the library management of people's hair follicle stem cells in, namely complete the structure in people's hair follicle stem cells storehouse.
2. the construction process in people's hair follicle stem cells storehouse as described in claim 1, it is characterized in that, described hair follicle stem cells nutrient solution be containing support the sugar of stem cell growth, amino acid, somatomedin, inorganic salt, trace element the DMEM/F12 nutrient solution of commerical prod, described DMEM/F12 nutrient solution contains 10% foetal calf serum, bFGF, 100u/mL are dual anti-for 10ng/mL Prostatropin.
3. the construction process in people's hair follicle stem cells storehouse as claimed in claim 1, is characterized in that, the aqueous solution of described equilibrated phosphate buffered soln mainly containing inorganic salt, is selected from PBS salt buffer solution, D-Hanks salt buffer solution, or SPB salt buffer solution.
4. the construction process in people's hair follicle stem cells storehouse as claimed in claim 1; it is characterized in that, described stem cell cryopreserving liquid contains cell freezing protective material, serum and stem cell minimum medium; wherein cell freezing protective material, the proportioning of serum and stem cell minimum medium is 1:2:7.
5. the construction process in people's hair follicle stem cells storehouse as claimed in claim 1, is characterized in that, the method for described program mode cooling is: preserve 40-60min for 4 DEG C, and preserve 1-2h for-20 DEG C ,-80 DEG C are spent the night, and are finally deposited into-196 DEG C of liquid nitrogen; Or be directly be placed in Programmed cryopreservation box, put into-80 DEG C of refrigerator overnight, within second day, forward in the liquid nitrogen of-196 DEG C.
6. the construction process in people's hair follicle stem cells storehouse as claimed in claim 1, it is characterized in that, described step 2) and the digestive process of step 3) also comprise: digest 3-5 minute under 37 DEG C of conditions after, examine under a microscope the digestion situation of cell, with the light beating cell culture apparatus of have gentle hands to help cell detachment.
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