CN115141795B - Preparation method of skin stem cells - Google Patents
Preparation method of skin stem cells Download PDFInfo
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Abstract
The invention relates to a preparation method of skin stem cells, and belongs to the technical field of biology. After skin tissue is cleaned, disinfected and centrifuged, the skin tissue is digested in a 37 ℃ incubator by using 1% collagenase IV and a complete culture medium mixed solution containing fetal calf serum, chitin, vitamin E and phytic acid, and then primary cell culture and subculture cell culture are sequentially carried out, so that skin stem cells are obtained. In the invention, a special culture medium and collagenase IV mixed solution are adopted in the skin tissue digestion process, and the skin tissue is digested for 12 to 36 hours in a 37 ℃ incubator, so that the complicated shaking table shaking step is eliminated, and the cost is reduced; the special culture medium is adopted for primary culture, and at least 2 million cells can be harvested after one T25 culture bottle is used for culturing for 6-10 days, so that the primary cell output time is greatly shortened, the primary cell production quantity is further improved, and a foundation is provided for mass production.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a preparation method of skin stem cells.
Background
Stem cells are cells with self-replication and multidirectional differentiation capabilities, can be continuously self-renewed, and can be differentiated into one or more cells constituting human tissues or organs under specific conditions, so that the tissues can recover the regeneration capability and restore the functions of the tissues. Researchers found a new adult stem cell under the dermis of human skin, which was named skin stem cell.
In today's society, high pressure and fast paced life present new challenges to the physical and mental health of people, loving mind, and all people, various human cultures throughout the history may consider certain physical features attractive, such as tall stature, clean skin, etc., so that other features may be ignored. However, as the human body organ physiological functions are changed and the functions are reduced with the increase of the years, the anti-aging requirements are increasingly enhanced, and as the research of regenerative medicine is continued deeply, the therapy of cell intervention is increasingly widely applied, namely, the body is supplemented with deficient cells, and the aged cells of the body are replaced so as to enable the body to be healthy and young, thereby inhibiting the aging and enabling the majority of friends to regain the beauty of youth and health.
With age, skin may develop aging phenomena such as sagging, dry and rough skin, reduced elasticity, increased wrinkles, etc., which are all associated with reduced stem cell numbers and reduced or abnormal secretory synthesis. Human skin stem cells are the most important cells in the dermis reticulation layer of skin, and are one of the main repair cells after skin aging and cell damage. It not only can promote migration, proliferation and differentiation of epidermal cells, but also can secrete a large amount of collagen, elastin and various cell repair factors, and has strong self-renewal capacity, so that aged skin can be repaired. Skin stem cells are important cells for maintaining skin elasticity and toughness, and play an important role in resisting skin aging due to their unique biological characteristics.
The stem cells developed and cultured through the process are injected into the skin by extracting the surface tissues of the human body, the collagen is secreted, the collagen structure is thickened and compact, the skin elasticity is enhanced, and the filling of skin pits, fine lines and damaged parts is realized, so that the anti-aging requirement of people can be met.
Skin is the largest organ of human body, and the stem cells are easy to obtain and have strong proliferation and division capacity, so that the culture of the skin stem cells has important significance for research and exploration of the connective tissue related field. The method for exploring and establishing the skin stem cell culture provides a reliable scientific basis for the application of the skin stem cells in clinical medicine.
Patent publication No. CN105969719A discloses a method for preparing skin fibroblast, firstly cutting human abdominal skin, placing in PBS containing ampicillin and streptomycin sulfate for rinsing, cutting the rinsed skin into small pieces, adding into DMEM containing collagenase and hyaluronidase, standing overnight at 4 ℃, then adding PBS digestive juice containing pancreatin and EDTA, centrifugally collecting cells, eluting with DMEM, then opening and culturing for 48 hours, rinsing with PBS without calcium and magnesium ions after the cultured cells grow into a monolayer, adding PBS digestive juice containing pancreatin and EDTA for digestion, adding a-MEM culture solution containing human autologous serum for blowing to terminate digestion, then subculturing for 48 hours, and finally centrifuging to obtain skin stem cells. The digestion process disclosed by the invention is divided into two steps, wherein the first step is to be placed overnight at the temperature of 4 ℃, and the digestion liquid is DMEM containing collagenase and hyaluronidase; the second digest was PBS digest containing pancreatin and EDTA.
Patent CN110438067a discloses a human skin fibroblast and a preparation method thereof, the preparation method comprises the following steps: pretreatment: collecting skin tissue behind the ear, separating dermis layer tissue, cleaning the dermis layer tissue with tissue cleaning liquid, and cutting into tissue fragments; first digestion: mixing and digesting the tissue fragments with the tissue digestion liquid I, centrifuging after digestion to remove supernatant, and reserving sediment; wherein, the tissue digestion solution I is normal saline containing neutral protease and DNase, and the second digestion is carried out: mixing the precipitate remained after the first digestion with tissue digestion solution II for digestion, and adding trypsin for continuous digestion; wherein the tissue digestion solution II is DNEM culture medium containing type I collagenase and DNase; terminating digestion: adding stop solution to stop digestion, sieving to remove tissue block, centrifuging, removing supernatant, and retaining precipitate; culturing: resuspension and precipitation with selective culture medium, and sub-packaging and culturing. Although the type I collagenase is disclosed in the second digestion process, the digestion method is I, and shake digestion is performed for 50-70 min at 37+ -1deg.C.
Patent publication number CN113621555a discloses a preparation method of skin fibroblasts, comprising the following steps: s01, specimen processing: treating skin specimen, and removing adipose tissue and connective tissue; s02, separating: treating the treated specimen with neutral protease to remove epidermis and obtain dermis tissue; s03, digestion: digesting the dermis tissue by using pancreatin to obtain tissue fluid; s04, centrifuging: centrifuging the tissue fluid, and removing the supernatant to obtain a cell suspension; s05, culturing: transferring the cell suspension to a culture dish, adding a culture solution, and culturing; s06, passage: culturing until the fusion degree is 75-85%, and subculturing. The method has the advantages of simple steps, difficult pollution, high cell survival rate, good purification effect and less cell loss. The invention does not disclose a technical scheme for digesting skin tissues overnight in a collagenase incubator at 37 ℃.
Patent publication No. CN111424010A discloses a preparation method of a skin fibroblast preparation for facial lifting, which mainly comprises the steps of obtaining and culturing autologous adipose-derived stem cells and preparing the skin stem cells.
Disclosure of Invention
The invention aims to provide a preparation method of skin stem cells, which comprises the steps of washing and disinfecting skin tissues, centrifuging, digesting the skin tissues in a 37 ℃ incubator by using a mixed solution of 1% collagenase IV and a complete culture medium containing fetal calf serum, chitin, vitamin E and phytic acid, and then sequentially carrying out primary cell culture and subculture to obtain the skin stem cells. In the invention, a special culture medium and collagenase IV mixed solution are adopted in the skin tissue digestion process, and the mixture is digested for 12-36 hours in a 37 ℃ incubator, so that the complicated shaking table shaking step is eliminated, and the cost is reduced; the special culture medium is adopted for primary culture, and at least 2 million cells can be harvested after one T25 culture bottle is used for culturing for 6-10 days, so that the primary cell output time is greatly shortened, the primary cell production quantity is further improved, and a foundation is provided for mass production.
The aim of the invention can be achieved by the following technical scheme:
a method for preparing skin stem cells, comprising the steps of:
(1) Pretreatment: cleaning and sterilizing skin tissues, then shearing the skin tissues into tissue homogenate blocks, centrifuging and discarding the supernatant to obtain dermis tissues;
(2) Digestion: digestion of dermal tissue using complete medium and 1% collagenase IV;
(3) Primary culture: the dermal tissue is digested for 12 to 36 hours, DPBS is used for cleaning and centrifuging to collect cells, a complete culture medium is used for primary cell culture for 6 to 10 days, and when the cell confluence reaches more than 80 percent, tissue blocks are digested and filtered to obtain cell suspension;
(4) Subculture: and (3) centrifuging the cell suspension by using DPBS, then carrying out cell subculture, and digesting and centrifuging to obtain the skin stem cells when the cell confluency reaches more than 85%.
As a preferable technical scheme of the invention, the cleaning and disinfecting step (1) refers to soaking, cleaning and disinfecting by using 1% of the green streptomycin and 0.9% of the sodium chloride injection in sequence.
As a preferred embodiment of the present invention, the complete medium in step (2) and step (3) comprises basal medium and fetal bovine serum, chitin, vitamin E, phytic acid.
As a more preferable technical scheme of the invention, the proportion of the basal medium, the fetal bovine serum, the chitin, the vitamin E and the phytic acid in the complete culture medium is as follows: 1ml of the mixture is 0.02-0.04 ml of the mixture is 20-50 ug of the mixture is 25-65 ug of the mixture is 8-18 ug of the mixture.
As a preferred embodiment of the present invention, the digestion conditions in step (2) are 5% CO at 37 DEG C 2 Placing in an incubator for 12-36 h.
As a preferable technical scheme of the invention, the primary culture specific method in the step (3) comprises the following steps: and (3) digesting the dermal tissue for 12-36 hours, cleaning by using DPBS, centrifuging to collect cells, performing primary culture on the cells for 6-10 days by using a complete culture medium, and digesting and filtering tissue blocks by using 0.125% trypsin digestion liquid when the confluency of the cells reaches more than 80%, thereby obtaining cell suspension.
As a preferred embodiment of the present invention, the step (3) of filtering the tissue mass means filtering the tissue mass through a 100 μm cell screen to obtain a cell suspension.
In a preferred embodiment of the present invention, the digestion solution for digestion in the step (4) is a 0.125% trypsin digestion solution.
As a preferred embodiment of the present invention, the digestion completion index in step (4) is: observing digestion condition under microscope, most cells are changed into round shape by shuttle, and the side wall of the culture flask is rapidly flapped until most cells fall off.
As a preferred embodiment of the present invention, the subculture in the step (4) means passage to the generation P2.
The invention has the beneficial effects that:
(1) The complete culture medium consists of a basic culture medium, fetal bovine serum, chitin, vitamin E and phytic acid, and the culture medium can increase the stability of skin stem cells, so that skin tissues can be stably digested in a culture box with 1% of collagenase IV and at 37 ℃; wherein, the vitamin E and the phytic acid can synergistically improve the proliferation speed of skin stem cells, and can greatly shorten the primary cell output time.
(2) The complete culture medium is adopted in the skin tissue digestion process, so that the skin tissue can be stably digested in a culture box with 1% of collagenase IV and 37 ℃, the complicated shaking table vibration step is eliminated, and the cost is reduced;
(3) The invention adopts the complete culture medium to carry out primary culture, and at least 2 million cells can be harvested after one T25 culture bottle is used for culturing for 6-10 days, thereby greatly shortening the primary cell output time, further improving the primary cell production quantity and providing a foundation for batch production.
Drawings
The present invention is further described below with reference to the accompanying drawings for the convenience of understanding by those skilled in the art.
FIG. 1 is a culture picture on day 1 of primary culture in example 1;
FIG. 2 is a culture picture on day 2 of primary culture in example 1;
FIG. 3 is a culture picture on day 4 of primary culture in example 1;
FIG. 4 is a culture picture on day 6 of primary culture in example 1;
FIG. 5 is a photograph showing the cultivation of P1 cells in 3T 75 flasks in example 1;
FIG. 6 is a photograph showing the cultivation of P2 cells in 3T 75 flasks in example 1;
FIG. 7 is a graph showing the results of the flow cytometer in example 1;
FIG. 8 is a graph showing the result of alizarin red dye solution staining in example 1;
FIG. 9 is a graph showing the result of dyeing with the oil red O dye liquor in example 1;
FIG. 10 is a graph showing the results of the blue dye liquor dyeing of example 1;
FIG. 11 is a culture picture on day 1 of primary culture in example 2;
FIG. 12 is a culture picture on day 2 of primary culture in example 2;
FIG. 13 is a photograph showing the culture of example 2 on day 4 of primary culture;
FIG. 14 is a photograph showing the culture of example 2 on day 6 of primary culture;
FIG. 15 is a photograph showing the cultivation of P1 cells in 3T 75 flasks in example 2;
FIG. 16 is a photograph showing the cultivation of P2 cells in 3T 75 flasks as described in example 2;
FIG. 17 is a culture picture on day 1 of primary culture in example 3;
FIG. 18 is a photograph showing the culture of example 3 on day 2 of primary culture;
FIG. 19 is a photograph showing the culture of example 3 on day 4 of primary culture;
FIG. 20 is a culture picture on day 5 of primary culture in example 3;
FIG. 21 is a photograph showing the primary culture of example 3 on day 7;
FIG. 22 is a photograph showing the cultured P1 cells in 3T 75 flasks in example 3;
FIG. 23 is a photograph showing the cultivation of P2 cells in 3T 75 flasks in example 3.
Detailed Description
In order to further describe the technical means and effects adopted by the present invention for achieving the intended purpose, the following detailed description will refer to the specific implementation, structure, characteristics and effects according to the present invention with reference to the accompanying drawings and preferred embodiments.
Example 1
(1) Normal skin was removed with sterilized forceps and placed to contain 15ml1Washing the skin with a first culture dish of a solution of% green streptomycin (0.9% sodium chloride injection), immersing the skin in a second culture dish containing 20ml of a solution of 0.9% sodium chloride injection for 1min to soften and disinfect, taking out the skin with hemostat, washing the skin with a third culture dish, taking out the skin with hemostat, cutting the skin with tissue scissors, and cutting the skin into 1-3 mm 3 Transferring the sheared skin to a 10ml centrifuge tube, adding 10ml of 0.9% sodium chloride injection, gently shaking, centrifuging 300g, and 10min;
(2) After centrifugation, the supernatant was discarded, the pellet was transferred to a T25 flask, 4ml of complete medium and 0.5ml of 1% collagenase IV were added, and the flask was placed at 37℃with 5% CO 2 Placing in an incubator for 20 hours, wherein the complete culture medium consists of a basic culture medium, fetal calf serum, chitin, vitamin E and phytic acid, and the proportion is as follows: 1ml:0.03ml:36ug:41ug:12ug;
(3) Taking out the T25 culture flask from the incubator, lightly shaking the culture flask, blowing the skin tissue suspension in the culture flask by a pipette to uniformly disperse tissue blocks, transferring the skin tissues and cells into a 10ml centrifuge tube, lightly flushing the culture flask with 1-2 ml DPBS for 1-2 times, transferring the washing solution into the 10ml centrifuge tube, and centrifuging for 500g and 5 min. Removing the supernatant, adding 1ml of the complete culture medium in the step (2) into a 10ml centrifuge tube, gently blowing tissue blocks and cells, inoculating the tissue blocks and the cells into a T25 culture flask, and adding 5ml/T25 of the complete culture medium in the step (2); culturing for 24h, sucking the old culture medium, adding 5ml serum-free culture medium/T25, photographing, and recording the adherence condition, wherein the culture picture is shown in figure 1; continuously culturing for 24 hours, photographing and recording the adherence condition, wherein a culturing picture is shown in figure 2; continuing to culture for 48 hours, sucking the old culture medium, adding 5ml of serum-free culture medium/T25, photographing and recording the growth condition, wherein the culture picture is shown in figure 3; observing cells under a microscope, culturing on the 6 th day, photographing and recording growth conditions, wherein a culture picture is shown in figure 4, the confluency reaches 80%, and digesting and harvesting; adding 1.5ml/T25 of 0.125% trypsin digestion solution, adding 5ml DPBS into each bottle after 2min to stop digestion, and repeatedly blowing the bottle bottom until most of cells fall off; preparing 50ml centrifuge tubes in a biosafety cabinet, opening the cover, placing the centrifuge tubes on a centrifuge tube rack at intervals, taking a 100 mu m cell screen which is packaged without damage into the biosafety cabinet, lightly pressing to confirm whether the biosafety cabinet is airtight, tearing the edge of a packaging bag, expanding a shearing opening and enabling the shearing opening to be upward, clamping a plastic protruding end of the filter screen by forceps in the biosafety cabinet, placing the plastic protruding end of the filter screen on the opened centrifuge tubes, sucking a cell suspension of a T25 culture bottle by using a pipette, filtering out tissue blocks by using the 100 mu m cell screen placed on the opened centrifuge tubes, collecting cells passing through the screen into the centrifuge tubes below the screen, adding 10ml DPBS into the bottle, resuspending the tissue blocks and cell precipitates, sucking the suspension, filtering out the tissue blocks by using the 100 mu m cells placed on the opened centrifuge tubes, and collecting the cells passing through the screen into the centrifuge tubes below the screen; balancing a 50ml centrifuge tube filled with cell suspension after filtration, putting the cell suspension into a centrifuge, centrifuging for 5min, discarding the centrifugal washing supernatant, re-suspending cells by using 5ml DPBS, combining the re-suspended cell suspension into 110 ml centrifuge tube, fixing the volume to 10ml, blowing uniformly, taking 0.2ml of cell suspension into an EP tube, measuring the total number and the activity of cells by a countStar cell counter, and calculating the total number of the living cells, wherein the obtained results are shown in Table 2;
(4) Centrifuging the constant volume cell suspension at 4000-8000 pieces/cm for 500g,5min 2 Inoculating the cells into 3T 75 culture flasks at a density, subculturing the skin stem cells for 72-84 hours, observing the cells under a microscope, obtaining P1 generation cells, recording the growth condition of the cells, and taking 3 cell pictures as shown in figure 5; preheating 0.125% trypsin digestion solution at 37 ℃ in a water bath kettle, rewarming the complete culture medium at normal temperature, sucking and discarding the culture medium in T75 culture flasks, adding 5ml DPBS into each T75 culture flask, cleaning for 1 time, adding 3ml/T75 of 0.125% g/ml trypsin digestion solution, observing digestion conditions under a microscope, changing most of cells into a round shape by shuttles, rapidly beating the side wall of the culture flask until most of cells fall off, adding 7ml PBS into each flask to dilute the digestion process after digestion is finished, repeatedly blowing the bottom of the flask until most of the cells fall off, transferring into a 10ml centrifuge tube, centrifuging and washing 500g for 5min, discarding the supernatant after centrifuging and washing, and repeating the stepsAfter the cells were resuspended with 10ml of DPBS for a 10ml centrifuge tube and blown uniformly, the total number of cells and the viability were measured by a CountStar cell counter, and the total number of viable cells was calculated, and the results are shown in Table 3; p2 generation cells were harvested by the same method as described above, cell growth was recorded and 3 cell pictures were taken as shown in FIG. 6, and the total number of cells and the viability were also measured by a CountStar cell counter and the total number of viable cells was calculated, and the results are shown in Table 4;
(5) Collecting the obtained P2 generation cell suspension, centrifuging at 1000rpm for 10min, removing supernatant, washing cell precipitate with 4 deg.C precooled 0.01mol/l PBS for 2 times at 1000rpm for 10min, re-suspending cell precipitate with 200ul PBS, collecting 1×10 6 Individual cells were resuspended in 0.1ml of pre-chilled 0.01mol/l PBS, fluorescent antibody was added in the dark, incubated at 4 ℃ for 30min in the dark, 2mL PBS,l000 rpm,10min was added, cells were washed to remove unbound antibody, supernatant was removed, cell pellet was resuspended in PBS, and detection was performed by flow cytometry, as shown in fig. 7, and the detection results showed: CD73/CD90/CD105 > 99%, CD19/CD16b/CD34/CD45/HLA-DR < 1%, indicating that the harvested P2 generation cells are skin stem cells;
(6) Continuously subculturing the P2 generation cells to P4 generation cells, and carrying out three-line induced differentiation:
a: osteogenic induced differentiation: when the cell fusion degree reaches 70% -80%, discarding the culture supernatant, adding 1ml of osteogenesis inducing complete medium into each hole of the sample group, adding 1ml of osteogenesis inducing complete medium into each hole of the control group, putting into 37 ℃, and adding 5% CO 2 Culturing in an incubator, discarding the in-hole osteogenesis inducing culture medium every 2-3 days, adding 1ml of fresh osteogenesis inducing culture medium, co-culturing for 32-36 days, and then carrying out alizarin red dye liquor, wherein the dyeing result is shown in figure 8, and the result shows that skin stem cells have osteogenesis capability;
b: adipogenic induced differentiation: when the cell fusion degree reaches 100%, the culture supernatant is discarded, 1ml of complete adipogenic induction culture medium ADP1 is added into each hole of the sample group, 1ml of complete adipogenic induction culture medium ADP1 is added into each hole of the control group, and the mixture is put into 37 ℃ and 5% CO 2 Culturing in incubator, discarding in-well lipid-forming induction culture medium ADP1 every 2-3 days, and adding 1ml fresh lipid-forming induction culture medium ADP2, after 2-3 times of alternate culture of the two culture mediums, continuing to culture by using an ADP2 culture medium, and performing oil red O dye solution after co-culture for 28-32 days, wherein the dyeing result is shown in figure 9, and the result shows that the skin stem cells have lipid-forming capability;
c: cartilage-forming induced differentiation: culturing in 15ml centrifuge tube, changing fresh complete chondrogenic induction culture medium every 2-3 days, sucking old culture medium, adding 0.5ml fresh complete chondrogenic induction culture medium into each tube, slightly stirring centrifuge tube after changing liquid to suspend cell pellet, placing at 37deg.C, and 5% CO 2 Culturing in an incubator, performing slice and alisxin blue staining after 18-21 days of induction culture, wherein the staining results are shown in figure 10, and the results show that the skin stem cells have the cartilage forming capacity.
Example 2
(1) Taking out skin with sterilized forceps, placing into a first culture dish containing 15ml of 1% green streptomycin solution (0.9% sodium chloride injection), washing off blood in skin, soaking skin in a second culture dish containing about 20ml of 0.9% sodium chloride injection for 1min for softening and sterilizing, taking out with hemostatic forceps, placing into a third culture dish for washing, taking out with hemostatic forceps, placing into culture dish, shearing with tissue scissors, shearing into pieces of 1-3 mm 3 Transferring the sheared skin to a 10ml centrifuge tube, adding 10ml of 0.9% sodium chloride injection, gently shaking, centrifuging 300g, and 10min;
(2) After centrifugation, the supernatant was discarded, the pellet was transferred to a T25 flask, 6ml of complete medium and 0.5ml of 1% collagenase IV were added, and the flask was placed at 37℃with 5% CO 2 Placing in an incubator for 18 hours, wherein the complete culture medium consists of a basic culture medium, fetal calf serum, chitin, vitamin E and phytic acid, and the proportion is as follows: 1ml:0.03ml:36ug:41ug:12ug;
(3) Taking out the T25 culture flask from the incubator, lightly shaking the culture flask, blowing the skin tissue suspension in the culture flask by a pipette to uniformly disperse tissue blocks, transferring the skin tissues and cells into a 10ml centrifuge tube, lightly flushing the culture flask with 1-2 ml DPBS for 1-2 times, transferring the washing solution into the 10ml centrifuge tube, and centrifuging for 500g and 5 min. Removing the supernatant, adding 1ml of the complete culture medium in the step (2) into a 10ml centrifuge tube, gently blowing tissue blocks and cells, inoculating the tissue blocks and the cells into a T25 culture flask, and adding 5ml/T25 of the complete culture medium in the step (2); culturing for 36h, sucking off old culture medium, adding 5ml serum-free culture medium/T25, photographing, and recording adherence condition, wherein the culture picture is shown in figure 11; continuously culturing for 48 hours, photographing and recording the adherence condition, wherein a culturing picture is shown in figure 12; continuing to culture for 96 hours, sucking the old culture medium, adding 5ml of serum-free culture medium/T25, photographing and recording the growth condition, wherein a culture picture is shown in figure 13; observing cells under a microscope, culturing on the 6 th day, photographing and recording growth conditions, wherein a culture picture is shown in fig. 14, the confluency reaches 80%, and digesting and harvesting; adding 1.5ml/T25 of 0.125% trypsin digestion solution, adding 5ml DPBS into each bottle after 2min to stop digestion, and repeatedly blowing the bottle bottom until most of cells fall off; preparing 50ml centrifuge tubes in a biosafety cabinet, opening the cover, placing the centrifuge tubes on a centrifuge tube rack at intervals, taking a 100 mu m cell screen which is packaged without damage into the biosafety cabinet, lightly pressing to confirm whether the biosafety cabinet is airtight, tearing the edge of a packaging bag, expanding a shearing opening and enabling the shearing opening to be upward, clamping a plastic protruding end of the filter screen by forceps in the biosafety cabinet, placing the plastic protruding end of the filter screen on the opened centrifuge tubes, sucking a cell suspension of a T25 culture bottle by using a pipette, filtering out tissue blocks by using the 100 mu m cell screen placed on the opened centrifuge tubes, collecting cells passing through the screen into the centrifuge tubes below the screen, adding 10ml DPBS into the bottle, resuspending the tissue blocks and cell precipitates, sucking the suspension, filtering out the tissue blocks by using the 100 mu m cells placed on the opened centrifuge tubes, and collecting the cells passing through the screen into the centrifuge tubes below the screen; balancing a 50ml centrifuge tube filled with cell suspension after filtration, putting the cell suspension into a centrifuge, centrifuging for 5min, discarding the centrifugal washing supernatant, re-suspending cells by using 5ml DPBS, combining the re-suspended cell suspension into 110 ml centrifuge tube, fixing the volume to 10ml, blowing uniformly, taking 0.2ml of cell suspension into an EP tube, measuring the total number and the activity of cells by a countStar cell counter, and calculating the total number of the living cells, wherein the obtained results are shown in Table 2;
(4) Centrifuging the constant volume cell suspension at 4000-8000 pieces/cm for 500g and 5min 2 Inoculating the cells into 3T 75 culture flasks at a density, subculturing the skin stem cells to 72-96 h, observing the cells under a microscope until the cell confluence reaches more than 85%, harvesting P1 generation cells, recording the growth condition of the cells, and taking 3 cell pictures as shown in figure 15; preheating 0.125% trypsin digestion solution at 37 ℃ in a water bath kettle, rewarming a complete culture medium at normal temperature, sucking and discarding the culture medium in T75 culture flasks, adding 5ml DPBS into each T75 culture flask, cleaning for 1 time, adding 3ml/T75 of 0.025% g/ml trypsin digestion solution, observing digestion conditions under a microscope, enabling most of cells to be changed into a round shape by shuttles, rapidly beating the side wall of the culture flask until most of cells fall off, adding 7ml PBS into each flask to dilute the digestion process after digestion is finished, repeatedly blowing the bottom of the flask until most of the cells fall off, transferring the cells into a 10ml centrifuge tube, centrifugally washing 500g and 5min, discarding centrifugally washed supernatant, re-suspending cells by using 10ml DPBS for each 10ml centrifuge tube, measuring the total number of cells by a CountStar cell counter after blowing uniformly, and calculating the total number of living cells, wherein the results are shown in Table 3; p2-generation cells were harvested by the same method as described above, cell growth was recorded and 3 cell pictures were taken as shown in FIG. 16, and the total number of cells, the viability was measured by the CountStar cytometer and the total number of viable cells was calculated as shown in Table 4.
Example 3
(1) The dermatitis skin is taken out by sterilized forceps and placed in a first culture dish containing 15ml of 1% green streptomycin solution (prepared by 0.9% sodium chloride injection), blood in the skin is washed off, then the skin is placed in a second culture dish containing about 20ml of 0.9% sodium chloride injection for softening and sterilizing, the skin is taken out by hemostat and placed in a third culture dish for washing, the skin is taken out by hemostat and placed in the culture dish for shearing by tissue scissors, and the skin is sheared into 1-3 mm 3 Transferring the sheared skin to a 10ml centrifuge tube, adding 10ml of 0.9% sodium chloride injection, gently shaking, centrifuging 300g, and 10min;
(2) After centrifugation, the supernatant was discarded and the pellet was transferred to a T25 flaskAdding 5ml complete culture medium and 0.5ml 1% collagenase IV, standing at 37deg.C, 5% CO 2 In the incubator, the complete culture medium is placed for 15 hours, and consists of basic culture medium, fetal calf serum, chitin, vitamin E and phytic acid, wherein the proportion is as follows: 1ml:0.03ml:36ug:41ug:12ug;
(3) Taking out the T25 culture flask from the incubator, lightly shaking the culture flask, blowing the skin tissue suspension in the culture flask by a pipette to uniformly disperse tissue blocks, transferring the skin tissues and cells into a 10ml centrifuge tube, lightly flushing the culture flask with 1-2 ml DPBS for 1-2 times, transferring the washing solution into the 10ml centrifuge tube, and centrifuging for 500g and 5 min. Removing the supernatant, adding 1ml of the complete culture medium in the step (2) into a 10ml centrifuge tube, gently blowing tissue blocks and cells, inoculating the tissue blocks and the cells into a T25 culture flask, and adding 5ml/T25 of the complete culture medium in the step (2); culturing for 48h, sucking the old culture medium, adding 5ml serum-free culture medium/T25, photographing, and recording the adherence condition, wherein the culture picture is shown in figure 17; continuously culturing for 48 hours, photographing and recording the adherence condition, wherein a culturing picture is shown in figure 18; continuing to culture for 48 hours, sucking the old culture medium, adding 5ml of serum-free culture medium/T25, photographing and recording the growth condition, wherein a culture picture is shown in figure 19; continuously culturing for 48 hours, photographing and recording the adherence condition, wherein a culturing picture is shown in figure 20; observing cells under a microscope, culturing on the 7 th day, photographing and recording growth conditions, wherein a culture picture is shown in figure 21, the confluency reaches 80%, and digesting and harvesting; adding 1.5ml/T25 of 0.125% trypsin digestion solution, adding 5ml DPBS into each bottle after 2min to stop digestion, and repeatedly blowing the bottle bottom until most of cells fall off; preparing 50ml centrifuge tubes in a biosafety cabinet, opening the cover, placing the centrifuge tubes on a centrifuge tube rack at intervals, taking a 100 mu m cell screen which is packaged without damage into the biosafety cabinet, lightly pressing to confirm whether the biosafety cabinet is airtight, tearing the edge of a packaging bag, expanding a shearing opening and enabling the shearing opening to be upward, clamping a plastic protruding end of the filter screen by forceps in the biosafety cabinet, placing the plastic protruding end of the filter screen on the opened centrifuge tubes, sucking a cell suspension of a T25 culture bottle by using a pipette, filtering out tissue blocks by using the 100 mu m cell screen placed on the opened centrifuge tubes, collecting cells passing through the screen into the centrifuge tubes below the screen, adding 10ml DPBS into the bottle, resuspending the tissue blocks and cell precipitates, sucking the suspension, filtering out the tissue blocks by using the 100 mu m cells placed on the opened centrifuge tubes, and collecting the cells passing through the screen into the centrifuge tubes below the screen; balancing a 50ml centrifuge tube filled with cell suspension after filtration, putting the cell suspension into a centrifuge, centrifuging for 5min, discarding the centrifugal washing supernatant, re-suspending cells by using 5ml DPBS, combining the re-suspended cell suspension into 110 ml centrifuge tube, fixing the volume to 10ml, blowing uniformly, taking 0.2ml of cell suspension into an EP tube, measuring the total number and the activity of cells by a countStar cell counter, and calculating the total number of the living cells, wherein the obtained results are shown in Table 2;
(4) Centrifuging the constant volume cell suspension at 4000-8000 pieces/cm for 500g,5min 2 Inoculating the cells into 3T 75 culture flasks at a density, subculturing the skin stem cells for 60-84 hours, observing the cells under a microscope, obtaining P1 generation cells, recording the growth condition of the cells, and taking 3 cell pictures as shown in figure 22; preheating 0.125% trypsin digestion solution at 37 ℃ in a water bath kettle, rewarming a complete culture medium at normal temperature, sucking and discarding the culture medium in T75 culture flasks, adding 5ml DPBS into each T75 culture flask, cleaning for 1 time, adding 3ml/T75 of 0.025% g/ml trypsin digestion solution, observing digestion conditions under a microscope, enabling most of cells to be changed into a round shape by shuttles, rapidly beating the side wall of the culture flask until most of cells fall off, adding 7ml PBS into each flask to dilute the digestion process after digestion is finished, repeatedly blowing the bottom of the flask until most of the cells fall off, transferring the cells into a 10ml centrifuge tube, centrifugally washing 500g and 5min, discarding centrifugally washed supernatant, re-suspending cells by using 10ml DPBS for each 10ml centrifuge tube, measuring the total number of cells by a CountStar cell counter after blowing uniformly, and calculating the total number of living cells, wherein the results are shown in Table 3; p2-generation cells were harvested by the same method as described above, cell growth was recorded and 3 cell pictures were taken as shown in FIG. 23, and the total number of cells, the viability was also measured by a CountStar cytometer and the total number of viable cells was calculated as shown in Table 4.
Comparative example 1
(1) Normal skin was removed with sterilized forceps and placed until it contained 15ml of 1% green streptomycin solution (0.9% sodium chloride injection) is used for washing the blood in the skin, then the skin is soaked in a second culture dish containing about 20ml of 0.9% sodium chloride injection for 1min to soften and disinfect, the skin is taken out by a pair of hemostats and put into a third culture dish to be washed, the skin is taken out by a pair of hemostats and put into the culture dish to be sheared by a pair of tissue scissors, and the skin is sheared into 1-3 mm 3 Transferring the sheared skin to a 10ml centrifuge tube, adding 10ml of 0.9% sodium chloride injection, gently shaking, centrifuging 300g, and 10min;
(2) After centrifugation, the supernatant was discarded, the pellet was transferred to a T25 flask, 4ml of complete medium and 0.5ml of 1% collagenase IV were added, and the flask was placed at 37℃with 5% CO 2 Placing in an incubator for 23 hours, wherein the complete culture medium consists of a basic culture medium, fetal calf serum, chitin and vitamin E, and the proportion is as follows: 1ml:0.03ml:36ug:53ug;
(3) Taking out the T25 culture flask from the incubator, lightly shaking the culture flask, blowing the skin tissue suspension in the culture flask by a pipette to uniformly disperse tissue blocks, transferring the skin tissues and cells into a 10ml centrifuge tube, lightly flushing the culture flask with 1-2 ml DPBS for 1-2 times, transferring the washing solution into the 10ml centrifuge tube, and centrifuging for 500g and 5 min. Removing the supernatant, adding 1ml of the complete culture medium in the step (2) into a 10ml centrifuge tube, gently blowing tissue blocks and cells, inoculating the tissue blocks and the cells into a T25 culture flask, and adding 5ml/T25 of the complete culture medium in the step (2); culturing for 24h, sucking off old culture medium, adding 5ml serum-free culture medium/T25, continuously culturing for 72h, sucking off old culture medium, adding 5ml serum-free culture medium/T25, culturing for 11 days until the confluency reaches 80%, and digesting and harvesting; adding 1.5ml/T25 of 0.125% trypsin digestion solution, adding 5ml DPBS into each bottle after 2min to stop digestion, and repeatedly blowing the bottle bottom until most of cells fall off; preparing 50ml centrifuge tubes in a biosafety cabinet, opening the cover, placing the centrifuge tubes on a centrifuge tube rack at intervals, taking a 100 mu m cell screen which is packaged without damage into the biosafety cabinet, lightly pressing to confirm whether the biosafety cabinet is airtight, tearing the edge of a packaging bag, expanding a shearing opening and enabling the shearing opening to be upward, clamping a plastic protruding end of the filter screen by forceps in the biosafety cabinet, placing the plastic protruding end of the filter screen on the opened centrifuge tubes, sucking a cell suspension of a T25 culture bottle by using a pipette, filtering out tissue blocks by using the 100 mu m cell screen placed on the opened centrifuge tubes, collecting cells passing through the screen into the centrifuge tubes below the screen, adding 10ml DPBS into the bottle, resuspending the tissue blocks and cell precipitates, sucking the suspension, filtering out the tissue blocks by using the 100 mu m cells placed on the opened centrifuge tubes, and collecting the cells passing through the screen into the centrifuge tubes below the screen; after balancing a 50ml centrifuge tube filled with the cell suspension after filtration, putting the mixture into a centrifuge, centrifuging the mixture for 5min by 500g, and removing the supernatant after centrifugal washing to obtain skin stem cells.
Comparative example 2
(1) Taking out normal skin with sterilized forceps, placing into a first culture dish containing 15ml of 1% green streptomycin solution (0.9% sodium chloride injection), washing off blood in skin, soaking skin in a second culture dish containing about 20ml of 0.9% sodium chloride injection for 1min for softening and sterilizing, taking out with hemostat, placing into a third culture dish for washing, taking out with hemostat, placing into culture dish for shearing with tissue scissors, shearing into pieces of 1-3 mm 3 Transferring the sheared skin to a 10ml centrifuge tube, adding 10ml of 0.9% sodium chloride injection, gently shaking, centrifuging 300g, and 10min;
(2) After centrifugation, the supernatant was discarded, the pellet was transferred to a T25 flask, 4ml of complete medium and 0.5ml of 1% collagenase IV were added, and the flask was placed at 37℃with 5% CO 2 Placing in an incubator for 18 hours, wherein the complete culture medium consists of a basic culture medium, fetal calf serum, chitin and phytic acid, and the proportion is as follows: 1ml:0.03ml:36ug:53ug;
(3) Taking out the T25 culture flask from the incubator, lightly shaking the culture flask, blowing the skin tissue suspension in the culture flask by a pipette to uniformly disperse tissue blocks, transferring the skin tissues and cells into a 10ml centrifuge tube, lightly flushing the culture flask with 1-2 ml DPBS for 1-2 times, transferring the washing solution into the 10ml centrifuge tube, and centrifuging for 500g and 5 min. Removing the supernatant, adding 1ml of the complete culture medium in the step (2) into a 10ml centrifuge tube, gently blowing tissue blocks and cells, inoculating the tissue blocks and the cells into a T25 culture flask, and adding 5ml/T25 of the complete culture medium in the step (2); culturing for 24h, sucking off old culture medium, adding 5ml serum-free culture medium/T25, continuously culturing for 72h, sucking off old culture medium, adding 5ml serum-free culture medium/T25, culturing for 12 days until the confluency reaches 80%, and digesting and harvesting; adding 1.5ml/T25 of 0.125% trypsin digestion solution, adding 5ml DPBS into each bottle after 2min to stop digestion, and repeatedly blowing the bottle bottom until most of cells fall off; preparing 50ml centrifuge tubes in a biosafety cabinet, opening the cover, placing the centrifuge tubes on a centrifuge tube rack at intervals, taking a 100 mu m cell screen which is packaged without damage into the biosafety cabinet, lightly pressing to confirm whether the biosafety cabinet is airtight, tearing the edge of a packaging bag, expanding a shearing opening and enabling the shearing opening to be upward, clamping a plastic protruding end of the filter screen by forceps in the biosafety cabinet, placing the plastic protruding end of the filter screen on the opened centrifuge tubes, sucking a cell suspension of a T25 culture bottle by using a pipette, filtering out tissue blocks by using the 100 mu m cell screen placed on the opened centrifuge tubes, collecting cells passing through the screen into the centrifuge tubes below the screen, adding 10ml DPBS into the bottle, resuspending the tissue blocks and cell precipitates, sucking the suspension, filtering out the tissue blocks by using the 100 mu m cells placed on the opened centrifuge tubes, and collecting the cells passing through the screen into the centrifuge tubes below the screen; after balancing a 50ml centrifuge tube filled with the cell suspension after filtration, putting the mixture into a centrifuge, centrifuging the mixture for 5min by 500g, and removing the supernatant after centrifugal washing to obtain skin stem cells.
Wherein the culture days before harvest of primary culture of examples 1 to 3 and comparative examples 1 to 2 are shown in Table 1 below, it is apparent from the table that the complete medium used in the primary culture of comparative example 1 consists of basal medium, fetal bovine serum, chitin and vitamin E in the following proportions: 1ml:0.03ml:36ug:53ug, the complete medium used in the primary culture of comparative example 2 consisted of basal medium, fetal bovine serum, chitin and phytic acid in the proportions: 1ml:0.03ml:36ug:53ug, the complete medium used in example 1 consisted of basal medium, fetal bovine serum, chitin, vitamin E and phytic acid in the proportions: 1ml:0.03ml:36ug:41ug:12ug, the confluence reached 80% on day 6 in the primary culture of example 1, but 80% on day 9 in the primary culture of comparative example 1, and 80% on day 10 in the primary culture of comparative example 2. The vitamin E and the phytic acid in the complete culture medium can synergistically promote the proliferation speed of cells and shorten the production time of primary cultured cells.
TABLE 1 days of Primary culture Pre-harvest culture
| Project | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 |
| Days (d) | 6 | 6 | 7 | 11 | 12 |
TABLE 2 Primary cell culture count results
TABLE 3 P1 cell culture count results
TABLE 4 P2 cell culture count results
The present invention is not limited to the above embodiments, but is capable of modification and variation in detail, and other modifications and variations can be made by those skilled in the art without departing from the scope of the present invention.
Claims (6)
1. A method for preparing skin stem cells, comprising the steps of:
(1) Pretreatment: cleaning and sterilizing skin tissues, then shearing the skin tissues into tissue homogenate blocks, centrifuging and discarding the supernatant to obtain dermis tissues;
(2) Digestion: digestion of dermal tissue using complete medium and 1% collagenase IV;
(3) Primary culture: after the dermis tissue is digested for 12-36 hours, DPBS is used for cleaning and centrifuging to collect cells, a complete culture medium is used for primary cell culture, and when the cell confluence reaches more than 80%, tissue blocks are digested and filtered to obtain cell suspension;
(4) Subculture: centrifuging the cell suspension by using DPBS, then carrying out cell subculture, and digesting and centrifuging to obtain skin stem cells when the cell confluence reaches more than 85%;
the complete culture medium in the step (2) and the step (3) comprises basal culture medium, fetal bovine serum, chitin, vitamin E and phytic acid;
the ratio of the basic culture medium, the fetal bovine serum, the chitin, the vitamin E and the phytic acid in the complete culture medium is as follows: 1ml of the mixture is 0.02-0.04 ml of the mixture is 20-50 ug of the mixture is 25-65 ug of the mixture is 8-18 ug of the mixture;
the digestion conditions in step (2) are 5% CO at 37 DEG C 2 Placing in an incubator for 12-36 h;
the primary culture method in the step (3) comprises the following steps: after the dermis tissue is digested for 12-36 hours, DPBS is used for cleaning and centrifuging to collect cells, a complete culture medium is used for primary cell culture for 6-10 days, and when the cell confluence reaches more than 80%, 0.125% trypsin digestion solution is used for digesting and filtering tissue blocks to obtain cell suspension.
2. The method of claim 1, wherein the step (1) of washing and sterilizing is to soak, wash and sterilize with 1% of penicillin and 0.9% of sodium chloride injection.
3. The method of claim 1, wherein the step (3) of filtering the tissue mass comprises filtering the tissue mass through a 100 μm cell screen to obtain a cell suspension.
4. The method according to claim 1, wherein the digestion solution for digestion in the step (4) is 0.125% trypsin digestion solution.
5. The method of claim 1, wherein the digestion in step (4) is completed as follows: observing digestion condition under microscope, most cells are changed into round shape by shuttle, and the side wall of the culture flask is rapidly flapped until most cells fall off.
6. The method of claim 1, wherein the subculture in step (4) is passage to generation P2.
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