CN111808808A - Culture method of mesenchymal stem cells on acellular allogeneic dermal scaffold and application of mesenchymal stem cells - Google Patents
Culture method of mesenchymal stem cells on acellular allogeneic dermal scaffold and application of mesenchymal stem cells Download PDFInfo
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Abstract
The invention provides a culture method of mesenchymal stem cells on an acellular allogeneic dermal scaffold and application thereof, wherein the culture method comprises the following steps: s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells until the cells are 90% confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, performing shake culture at 37 ℃, and then performing static culture for 2-3 days, wherein the culture conditions are as follows: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity; the artificial skin prepared by the method further reduces the depth of the wound surface and provides relatively satisfactory healing effect.
Description
Technical Field
The invention belongs to the technical field of biological tissue organ repair, and particularly relates to a culture method of mesenchymal stem cells on an acellular allogeneic dermal scaffold and application of the mesenchymal stem cells.
Background
The normal skin is divided into three layers, namely a cuticle layer, a dermis layer and a subcutaneous tissue layer, wherein the dermis layer contains abundant nerves, blood vessels and the like, the dermis of the adult mammal cannot be spontaneously regenerated, when a transplanted and surviving dermal matrix is lacked, fibroblasts begin to synthesize an immature matrix, the matrix is reshaped to form scars, part of the scars shrink the skin to cause pain, part of the scars can also influence similar joint activities, and the long-term influence is exerted on the physical and psychological health of a patient; the existing acellular dermal matrix on the market is a dermal substitute obtained by specially treating allogenic dermis and removing cell components of the allogenic dermis, the biggest problem of allogenic transplantation is rejection reaction, and the cell components of the rejection reaction caused by skin transplantation, such as epidermal cells, fibroblasts in the dermis, endothelial cells and the like, are mainly used; the extracellular matrix proteins and collagen (i.e., decellularized dermis) which are not cellular components, have no immune activity and can permanently exist in the foreign host body, thereby effectively improving the skin contraction condition caused by scar formation.
The chronic wound surface has many reasons, except infection and common systemic diseases (such as diabetes), large-area skin defect is also the main reason of the chronic difficult-to-heal wound surface, the treatment time of the chronic wound surface is long, the physical and economic burden is caused to patients, and the social and psychological health of the patients is also seriously influenced, so the chronic wound surface is difficult to be used in plastic surgery and even the whole medical field; the existing clinical common repair method needs to damage the donor area, if the damaged area of the recipient area is large, the donor area may face the risks of large damage and poor healing again, in recent years, certain breakthroughs are made in promoting the research of chronic wounds, and stem cells provide a new idea for solving the problem.
Adipose-derived stem cells (ADSCs) are MSCs derived from mesoderm, can be differentiated in a multi-way mode across mesoderm, have autocrine and paracrine functions, can secrete various cytokines, relieve inflammatory reaction and promote angiogenesis, are different from embryonic stem cells and BMSCs, have wide acquisition ways of the ADSCs, can be acquired by local anesthesia, minimally invasive and other modes, and do not relate to ethical problems; therefore, researches show that the ADSCs can remarkably promote wound healing, and the researches prove that the ADSCs have anti-inflammatory and anti-fibrosis effects; however, according to the conventional cell therapy method, the ADSCs are directly injected into the wound tissue, but the effect is difficult to meet the requirement of a patient, and the possible reason is that the wound tissue is in a chronic inflammation state, the injected ADSCs cells are greatly affected by local environment, the survival rate is limited, and thus the effect of promoting wound healing is difficult to be exerted.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture method of mesenchymal stem cells on an acellular allogeneic dermal scaffold.
The specific technical scheme of the invention is as follows:
the invention provides a culture method of mesenchymal stem cells on an acellular allogeneic dermal scaffold, which specifically comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells until the cells are 70-90% confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, performing shake culture at 37 ℃, and then performing static culture for 2-3 days, wherein the culture conditions are as follows: the temperature is 37.0 + -0.5 deg.C, the concentration of CO2 is 5.0 + -0.2%, and the humidity is saturated humidity.
Further, the culture method specifically comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing with a complete culture medium until 70-90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is used as a basal medium, and human fibronectin with the concentration of 20-30 mu g/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 10-20ng/ml, ITS with the concentration of 0.5-1.5%, human serum albumin with the concentration of 4-6%, NEAA with the concentration of 0.5-1.5%, hydrocortisone with the concentration of 0.05-0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.05-0.15% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel, culturing by using a complete culture medium, firstly carrying out shake culture at 37 ℃ for 20-40min, controlling the rotating speed of a shaking table to be 40-60r/min, then stopping shake, and standing for continuous culture for 3-6 days, wherein the culture conditions are as follows: the temperature is 37.0 + -0.5 deg.C, the concentration of CO2 is 5.0 + -0.2%, and the humidity is saturated humidity.
Wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco; the acellular allogeneic dermal scaffold (ADM) is newly developed tissue engineering skin in recent years, has excellent compatibility with human tissues, does not have any rejection reaction, therefore, the acellular allogeneic dermal scaffold is often applied to organ reconstruction and tissue repair operations of plastic surgery, and obtains satisfactory operation effects, the acellular allogeneic dermal scaffold is a scaffold component of skin and can provide a good growth environment and a 'sheltering place' for skin cells, the research of the invention finds that ADSCs can survive well in the acellular allogeneic dermal scaffold and have corresponding biological activity, the ADSCs are combined with the acellular allogeneic dermal scaffold without immunogenicity, the formed human tissue engineering skin can not only meet the requirements, but also provide considerable tissue capacity, further reduce the depth of a wound surface and provide relatively satisfactory healing effects; the invention utilizes ADSCs to complement the deleted cell components for the acellular dermal matrix, and the mesenchymal stem cells and the acellular allogeneic dermal scaffold form tissue engineering skin which can promote the healing of the wound surface and accelerate the resolution of the graft inflammatory reaction.
Preferably, the cultivation method specifically comprises the steps of:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing the mesenchymal stem cells by using a complete culture medium until 90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 20 mu g/ml, basic fibroblast growth factor with the concentration of 5ng/ml, human epidermal growth factor with the concentration of 10ng/ml, ITS with the concentration of 0.5, human serum albumin with the concentration of 4 percent, NEAA with the concentration of 0.5 percent, hydrocortisone with the concentration of 0.05 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 percent are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel, culturing by using a complete culture medium, performing shake culture at 37 ℃ for 20min, controlling the rotating speed of a shaking table to be 40r/min, stopping shake, standing, and continuing to culture for 3 days, wherein the culture conditions are as follows: the temperature is 37.0 +/-0.5 ℃ and CO2The concentration is 5.0 +/-0.2%, and the humidity is saturated humidity.
Further, the mesenchymal stem cell comprises one of adipose mesenchymal stem cell, umbilical cord mesenchymal stem cell and endometrium mesenchymal stem cell.
Further, the mesenchymal stem cells are adipose mesenchymal stem cells, and the preparation method of the adipose mesenchymal stem cell suspension comprises the following steps:
(1) taking fat cells;
(2) placing the fat cells in a test tube, adding digestive juice with the volume same as that of the fat tissue into the test tube, wherein the digestive juice comprises trypsin-EDTA and 0.1% type I collagenase in a volume ratio of 1:1, sealing the test tube, placing the test tube in a constant-temperature shaking table at 37 ℃, and performing shock digestion at a speed of 180-;
(3) sucking the lower layer liquid in the centrifuge tube, transferring into a new centrifuge tube filled with complete culture medium to terminate digestion, sealing the centrifuge tube, centrifuging, and separating into 2 layers;
(4) sucking the supernatant in the new centrifugal tube by using a suction tube, removing the supernatant, adding a complete culture medium, and slightly blowing and beating to prepare cell suspension;
(5) the cell suspension was adjusted to 0.5X 106Inoculating to culture flask at density of one ml, placing at 37 deg.C and 5% CO2Culturing in an incubator; carrying out first liquid change after 12-24 hours, changing liquid every 3 days, and carrying out passage after cells grow to be fused;
(6) sucking out the old culture medium in the culture bottle, adding PBS for washing for 2-3 times, and adding digestive juice for digestion for 3-5min, wherein the digestive juice is trypsin-EDTA; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; and centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8.
Further, the adipocytes of step (1) are a fat aspirate directly obtained by liposuction.
Further, the method for obtaining adipocytes in step (1) comprises: squeezing adipose tissue into a centrifuge tube, sucking PBS buffer solution, adding into the centrifuge tube with tissue, using 12-16ml PBS buffer solution per gram of adipose tissue, blowing with a pipette for 10-20 times, standing for 0.5-1.5min, sucking out the liquid in the lower layer of tissue with the pipette, and repeating the above steps until the liquid is transparent and has no blood cells.
Further, the decellularized allodermal scaffold needs to be cut into square small pieces with the side length of 2cm before being placed in the ultra-low adsorption reaction vessel, and is washed for three times by using physiological saline.
The invention also provides an artificial skin prepared by the culture method.
The invention also provides the application of the artificial skin in the medical field of bionic materials.
The culture method provided by the invention has the following technical effects: the invention utilizes ADSCs to complement the lost cell components for the acellular dermal matrix, the mesenchymal stem cells and the acellular allodermal form tissue engineering skin which can promote the healing of the wound surface, accelerate the resolution of the graft inflammatory reaction and improve the treatment effect of the acellular dermal matrix, and the dermal matrix loaded with the cells can be used for deep burn wound surface repair, the filling function of plastic surgery, eyelid shaping and the like.
Drawings
FIG. 1 shows the results of measurement of anchorage rate of cells after 1 day of culture in each set of methods.
FIG. 2 shows the results of measuring the proliferation rate of cells after 1 day of culture in each group of methods.
FIG. 3 shows the effect of artificial skin or cells on the treatment of wounds in mice.
Detailed Description
Example 1
The embodiment provides a culture method of adipose-derived mesenchymal stem cells on an acellular allogeneic dermal scaffold, which comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing adipose-derived mesenchymal stem cells, culturing with complete culture medium until 90% of cells are confluent, collecting P3 generation cells, and making into mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 20 mu g/ml, basic fibroblast growth factor with the concentration of 5ng/ml, human epidermal growth factor with the concentration of 10ng/ml, ITS with the concentration of 0.5%, human serum albumin with the concentration of 4%, NEAA with the concentration of 0.5%, hydrocortisone with the concentration of 0.05 mu mol/L and beta-mercaptoethanol with the concentration of 0.05% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, firstly performing shake culture at 37 ℃ for 20min, controlling the rotating speed of a shaking table to be 40r/min, stopping shake, standing, and continuing culturing for 3 days (determining anchorage rate, proliferation multiple and adhesiveness after culturing for 1 day), wherein the culture conditions are as follows: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity;
wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco;
the preparation method of the adipose-derived mesenchymal stem cell suspension comprises the following steps:
(1) squeezing adipose tissues into a centrifuge tube, sucking PBS buffer solution, adding into the centrifuge tube with tissues, blowing 12ml PBS buffer solution per gram of adipose tissues with a suction tube for 10 times, standing for 0.5min, sucking out the liquid at the lower layer of the tissues with the suction tube, and repeating the steps until the sucked liquid is transparent and has no blood cells;
(2) placing 30ml of fat extraction liquid into a test tube, adding 30ml of digestive juice into the test tube, wherein the digestive juice comprises trypsin-EDTA and 0.1% type I collagenase in a volume ratio of 1:1, sealing the test tube, placing the test tube into a constant-temperature shaking table at 37 ℃, performing shake digestion at a speed of 180r/Min for 30Min, and dividing the test tube into 3 layers, wherein the upper layer is a yellow oily fat cell layer, the middle layer is a fat tissue layer, and the lower layer is liquid containing mononuclear cells;
(3) sucking the lower layer liquid in the centrifuge tube, transferring into a new centrifuge tube filled with complete culture medium to terminate digestion, sealing the centrifuge tube, centrifuging at 1500rpm for 10min, and separating into 2 layers;
(4) sucking the supernatant in the new centrifuge tube by a suction tube, removing the supernatant, adding 1ml of complete culture medium, and slightly blowing and beating for 15 times to prepare cell suspension;
(5) the cell suspension was adjusted to 0.5X 106Inoculating to culture flask at density of 20ml, placing at 37 deg.C and 5% CO2Culturing in an incubator; carrying out first liquid change after 12 hours, changing liquid every 3 days, and carrying out passage after the cells grow to be fused;
(6) sucking out the old culture medium in the culture bottle, adding PBS for washing for 2 times, adding 2ml of digestive juice for digestion for 3min, wherein the digestive juice is trypsin-EDTA; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8;
the acellular dermis used by the invention is a commercial domestic medical instrument commodity, is purchased from Beijing name of the last ruler of the Xia Dynasty Laifu, and the trypsin-EDTA of the application is purchased from Gibco.
Example 2
The embodiment provides a culture method of adipose-derived mesenchymal stem cells on an acellular allogeneic dermal scaffold, which comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing adipose-derived mesenchymal stem cells, culturing with complete culture medium until 90% of cells are confluent, collecting P3 generation cells, and making into mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 25 mu g/ml, basic fibroblast growth factor with the concentration of 10ng/ml, human epidermal growth factor with the concentration of 15ng/ml, ITS with the concentration of 1%, human serum albumin with the concentration of 5%, NEAA with the concentration of 1%, hydrocortisone with the concentration of 0.1 mu mol/L and beta-mercaptoethanol with the concentration of 0.1% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, firstly performing shake culture at 37 ℃ for 30min, controlling the rotating speed of a shaking table to be 50r/min, stopping shake, standing, and continuously culturing for 5 days (determining anchorage rate, proliferation multiple and adhesiveness after culturing for 1 day), wherein the culture conditions are as follows: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity;
wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco;
the preparation method of the adipose-derived mesenchymal stem cell suspension comprises the following steps:
(1) squeezing adipose tissues into a centrifuge tube, sucking PBS buffer solution, adding into the centrifuge tube with tissues, blowing 12ml PBS buffer solution per gram of adipose tissues with a suction tube for 10 times, standing for 0.5min, sucking out the liquid at the lower layer of the tissues with the suction tube, and repeating the steps until the sucked liquid is transparent and has no blood cells;
(2) placing 30ml of fat extraction liquid into a test tube, adding 30ml of digestive juice into the test tube, wherein the digestive juice comprises trypsin-EDTA and 0.1% type I collagenase in a volume ratio of 1:1, sealing the test tube, placing the test tube into a constant-temperature shaking table at 37 ℃, performing shake digestion at the speed of 190r/Min for 30Min, and dividing the test tube into 3 layers, wherein the upper layer is a yellow oily fat cell layer, the middle layer is a fat tissue layer, and the lower layer is liquid containing mononuclear cells;
(3) sucking the lower layer liquid in the centrifuge tube, transferring into a new centrifuge tube filled with complete culture medium to terminate digestion, sealing the centrifuge tube, centrifuging at 1500rpm for 10min, and separating into 2 layers;
(4) sucking the supernatant in the new centrifuge tube by a suction tube, removing the supernatant, adding 1ml of complete culture medium, and slightly blowing and beating for 15 times to prepare cell suspension;
(5) the cell suspension was adjusted to 0.5X 106Inoculating to culture flask at density of 20ml, placing at 37 deg.C and 5% CO2Culturing in an incubator; carrying out first liquid change after 18 hours, changing liquid every 3 days, and carrying out passage after the cells grow to be fused;
(6) sucking out the old culture medium in the culture bottle, adding PBS for washing for 2 times, adding 2ml of digestive juice for digestion for 4min, wherein the digestive juice is trypsin-EDTA; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8;
the acellular dermis used by the invention is a commercial domestic medical instrument commodity, is purchased from Beijing name of the last ruler of the Xia Dynasty Laifu, and the trypsin-EDTA of the application is purchased from Gibco.
Example 3
The embodiment provides a culture method of adipose-derived mesenchymal stem cells on an acellular allogeneic dermal scaffold, which comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing adipose-derived mesenchymal stem cells, culturing with complete culture medium until 90% of cells are confluent, collecting P3 generation cells, and making into mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 30 mu g/ml, basic fibroblast growth factor with the concentration of 15ng/ml, human epidermal growth factor with the concentration of 20ng/ml, ITS with the concentration of 1.5%, human serum albumin with the concentration of 6%, NEAA with the concentration of 1.5%, hydrocortisone with the concentration of 0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.15% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, firstly performing shake culture for 40min at 37 ℃, controlling the rotating speed of a shaking table to be 60r/min, stopping shake, standing, and continuously culturing for 6 days (determining anchorage rate, proliferation multiple and adhesiveness after culturing for 1 day), wherein the culture conditions are as follows: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity;
wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco;
the preparation method of the adipose-derived mesenchymal stem cell suspension comprises the following steps:
(1) squeezing adipose tissues into a centrifuge tube, sucking PBS buffer solution, adding into the centrifuge tube with tissues, blowing 16ml PBS buffer solution per gram of adipose tissues with a suction tube for 20 times, standing for 1.5min, sucking out the liquid in the lower layer of the tissues with the suction tube, and repeating the steps until the sucked liquid is transparent and has no blood cells;
(2) placing the fat cells in a test tube, adding digestive juice with the same volume as the fat tissue into the test tube, wherein the digestive juice comprises trypsin-EDTA and 0.1% type I collagenase in a volume ratio of 1:1, sealing the test tube, placing the test tube in a constant-temperature shaking table at 37 ℃, and performing shock digestion at the speed of 200r/Min to divide the test tube into 3 layers;
(3) sucking the lower layer liquid in the centrifuge tube, transferring into a new centrifuge tube filled with complete culture medium to terminate digestion, sealing the centrifuge tube, centrifuging, and separating into 2 layers;
(4) sucking the supernatant in the new centrifugal tube by using a suction tube, removing the supernatant, adding a complete culture medium, and slightly blowing and beating to prepare cell suspension;
(5) the cell suspension was adjusted to 0.5X 106Inoculating to culture flask at density of one ml, placing at 37 deg.C and 5% CO2Culturing in an incubator; carrying out first liquid change after 24 hours, changing liquid every 3 days, and carrying out passage after the cells grow to be fused;
(6) sucking out the old culture medium in the culture bottle, adding PBS for washing for 3 times, and adding digestive juice for digestion for 5min, wherein the digestive juice is trypsin-EDTA; gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain a single cell suspension; centrifuging and blowing off the single cell suspension, and carrying out passage according to the ratio of 1: 8;
the acellular dermis used by the invention is a commercial domestic medical instrument commodity, is purchased from Beijing name of the last ruler of the Xia Dynasty Laifu, and the trypsin-EDTA of the application is purchased from Gibco.
Comparative example 1
The control example provides a method for culturing adipose-derived stem cells, which comprises culturing adipose-derived stem cells in a polylysine culture dish (purchased from corning, model 431079), culturing by adherent culture, specifically inoculating 20ml of cell suspension into the culture dish, wherein the density of the cell suspension is 0.5 × 106Putting the culture dish into a carbon dioxide incubator at 37.0 +/-0.5 ℃ and 5.0 +/-0.2% of CO2Culturing under the conditions of concentration and saturated humidity for 5 days.
Comparative example 2
This comparative example provides a method for culturing mesenchymal stem cells, which is different from example 1 in that adipose-derived stem cells are replaced with mesenchymal stem cells; bone marrow mesenchymal stem cells were purchased from Guangzhou Seilara stem cell technologies, Inc.
Comparative example 3
The control example provides a method for culturing mesenchymal stem cells, which comprises the steps of culturing the mesenchymal stem cells by using a polylysine culture dish, wherein the polylysine culture dish is purchased from corning and has the model number of 431079, and the culturing method is adherent culture; specifically, 20ml of mesenchymal stem cell suspension is inoculated into a culture dish, and the density of the cell suspension is 0.5 multiplied by 106Putting the culture dish into a carbon dioxide incubator at 37.0 +/-0.5 ℃ and 5.0 +/-0.2% of CO2Culturing for 5 days under the conditions of concentration and saturated humidity; bone marrow mesenchymal stem cells were purchased from Guangzhou Seilara stem cell technologies, Inc.
Comparative example 4
The comparison example provides a culture method of adipose-derived mesenchymal stem cells on an acellular allogeneic dermal scaffold, which comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing the mesenchymal stem cells by using a complete culture medium until 90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 25 mu g/ml, basic fibroblast growth factor with the concentration of 10ng/ml, human epidermal growth factor with the concentration of 15ng/ml, ITS with the concentration of 1%, human serum albumin with the concentration of 5%, NEAA with the concentration of 1%, hydrocortisone with the concentration of 0.1 mu mol/L and beta-mercaptoethanol with the concentration of 0.1% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel, culturing for 5 days by using a complete culture medium, and performing shake culture at 37 ℃ for 30min at the rotating speed of a shaking table of 50r/min under the following culture conditions: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity;
wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco;
wherein, the preparation method of the adipose-derived mesenchymal stem cell suspension is the same as that of the embodiment 2;
the acellular dermis used by the invention is a commercial domestic medical instrument commodity, is purchased from Beijing name of the last ruler of the Xia Dynasty Laifu, and the trypsin-EDTA of the application is purchased from Gibco.
Comparative example 5
The present comparative example provides a method for culturing adipose-derived stem cells, which comprises the following steps:
the comparison example provides a culture method of adipose-derived mesenchymal stem cells on an acellular allogeneic dermal scaffold, which comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing the mesenchymal stem cells by using a complete culture medium until 90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 25 mu g/ml, basic fibroblast growth factor with the concentration of 10ng/ml, human epidermal growth factor with the concentration of 15ng/ml, ITS with the concentration of 1%, human serum albumin with the concentration of 5%, NEAA with the concentration of 1%, hydrocortisone with the concentration of 0.1 mu mol/L and beta-mercaptoethanol with the concentration of 0.1% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel, and performing static culture for 5 days by using a complete culture medium under the following culture conditions: the temperature is 37.0 +/-0.5 ℃, the concentration of CO2 is 5.0 +/-0.2 percent, and the humidity is saturated humidity;
wherein ITS (insulin-transferrin-selenium) comprises three components of insulin, transferrin and selenium solution, is purchased from Gibco, NEAA, Chinese name nonessential amino acid and is purchased from Gibco;
wherein, the preparation method of the adipose-derived mesenchymal stem cell suspension is the same as that of the embodiment 2;
the acellular dermis used by the invention is a commercial domestic medical instrument commodity, is purchased from Beijing name of the last ruler of the Xia Dynasty Laifu, and the trypsin-EDTA of the application is purchased from Gibco.
Test example 1 measurement of adherence rate, proliferation fold and adherence rate
The anchorage rate and proliferation rate of examples 1-3 and comparative examples 1-5 were measured, respectively, and examples 1-3 were measured after placing the decellularized allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction dish for static culture using a complete medium for one day in the S2 attachment step, comparative example 1 was performed by placing adipose-derived stem cells in a carbon dioxide incubator for one day, comparative example 2 was performed by placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel for static culture for one day in the step of S2 attachment, comparative example 3 was a method in which bone marrow mesenchymal stem cells were cultured in a carbon dioxide incubator for one day and then measured, comparative example 4 in the S2 attachment step, the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension were placed in an ultra-low adsorption reaction dish and shake-cultured for one day using a complete medium, and then measured; comparative example 5 the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension were placed in an ultra-low adsorption reaction dish for static culture for one day in the S2 attachment step, and the adhesion rate between adipose mesenchymal stem cells and the acellular dermis in the artificial skin prepared in examples 1-3 and comparative examples 2, 4, 5 was calculated, wherein the adhesion rate and the proliferation multiple were measured by counting under a microscope, and the adhesion rate was calculated by the formula of (%) adhesion rate [ (((number of seeded cells-number of non-adherent cells)/number of seeded cells) × 100%, and the results of measurement of the adhesion rate and proliferation multiple were shown in fig. 1 and 2, and the experimental examples in fig. 1 and 2 are examples.
As can be seen from fig. 1 and 2, the adhesion effect of examples 1 to 3 is higher than that of comparative examples 1 to 5, and on the other hand, compared with the methods of comparative examples 1 to 5, the proliferation rate of the ADSCs is higher when the adipose mesenchymal stem cells are co-cultured with the acellular allogeneic dermis, and it can be known by calculation that the ADSCs of examples 1 to 3 have good adhesion to the acellular allogeneic dermis with adhesion rates of 93.41%, 93.67% and 94.78%, respectively, whereas the adhesion rate of the BMSCs of comparative example 2 to the acellular allogeneic dermis is only 79.20%, the adhesion rate of the ADSCs to the acellular allogeneic dermis in the method of comparative example 4 is 80.40%, and the adhesion rate of the cs to the acellular allogeneic adsderma in the method of comparative example 5 is 82.40%, it can be seen that the adhesion rate of the adipose stem cells to the acellular allogeneic dermis in the method provided by the present invention is high, and when the adipose mesenchymal stem cells are replaced with the mesenchymal stem cells, the adhesion rate is remarkably reduced, and when the culture method of the cells is replaced by only shaking culture or only standing culture from the first shaking culture and then the standing culture, the adhesion rate is remarkably reduced.
Test example 2
Selecting 20C 57 mice 8-10 weeks old, and removing the injury of dorsal skin tissue after anesthesia by 1cm2Exposing the wound to the muscular layer, dividing 20 animals into injury control group, ADM treatment group, ADSCs treatment group and combination treatment group, each group comprises 5 animals, and implanting 1cm of wound into the dorsal skin of mice of ADM treatment group, combination treatment group and ADSCs treatment group2Blank ADM, 1cm2Artificial skin of example 1, adipose-derived mesenchymal stem cells of control example 1, ADSCs-treated groupAdipose-derived mesenchymal stem cells were administered by local subcutaneous injection in an amount of 4.3X 106The blank ADM group was the same as the acellular allogeneic dermal scaffold used in example 1, and then the wound was bandaged with a breathable dressing, and the wound control group was bandaged with a breathable dressing alone, and after one week, the change in the wound on the body surface of each animal group was observed, and the test results are shown in fig. 3.
As can be seen from FIG. 3, the combination treatment group using the artificial skin of example 1 exhibited significantly better maintenance of the body surface of the animals than the other 3 groups, and the wounds of the injured control group did not significantly change; the exudates of the wound on the back of ADM treated mice were essentially gone, but the wound was not completely closed; the exudation of the wounds on the back of the mice in the ADSCs treatment group is reduced, and the wounds are not completely closed; the wounds of the combined treatment group are completely recovered without exudates, and as can be seen from fig. 3, the artificial skin prepared by the method provided by the invention can promote the healing of the wound surface and further reduce the depth of the wound surface.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (10)
1. A culture method of mesenchymal stem cells on an acellular allogeneic dermal scaffold is characterized by comprising the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells until the cells are 70-90% confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultralow adsorption reaction vessel, culturing by using a complete culture medium, performing shake culture at 37 ℃, and then performing static culture for 2-3 days, wherein the culture conditions are as follows: the temperature is 37.0 + -0.5 deg.C, the concentration of CO2 is 5.0 + -0.2%, and the humidity is saturated humidity.
2. The method of culturing mesenchymal stem cells on an acellular allogeneic dermal scaffold according to claim 1, wherein the method of culturing comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing with a complete culture medium until 70-90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is used as a basal medium, and human fibronectin with the concentration of 20-30 mu g/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 10-20ng/ml, ITS with the concentration of 0.5-1.5%, human serum albumin with the concentration of 4-6%, NEAA with the concentration of 0.5-1.5%, hydrocortisone with the concentration of 0.05-0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.05-0.15% are added;
s2: attachment: placing the acellular allogeneic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel, culturing by using a complete culture medium, firstly carrying out shake culture at 37 ℃ for 20-40min, controlling the rotating speed of a shaking table to be 40-60r/min, then stopping shake, and standing for continuous culture for 3-6 days, wherein the culture conditions are as follows: the temperature is 37.0 + -0.5 deg.C, the concentration of CO2 is 5.0 + -0.2%, and the humidity is saturated humidity.
3. The method of culturing mesenchymal stem cells on an acellular allogeneic dermal scaffold according to claim 1, wherein the method of culturing comprises the following steps:
s1: preparing a mesenchymal stem cell suspension: subculturing the mesenchymal stem cells, culturing the mesenchymal stem cells by using a complete culture medium until 90% of the cells are confluent, collecting P3 generation cells, and preparing a mesenchymal stem cell suspension; the complete culture medium is as follows: DMEM/F12 is taken as a basal medium, and human fibronectin with the concentration of 20 mu g/ml, basic fibroblast growth factor with the concentration of 5ng/ml, human epidermal growth factor with the concentration of 10ng/ml, ITS with the concentration of 0.5, human serum albumin with the concentration of 4 percent, NEAA with the concentration of 0.5 percent, hydrocortisone with the concentration of 0.05 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 percent are added;
s2: attachment: placing the acellular allogenic dermal scaffold and the mesenchymal stem cell suspension in an ultra-low adsorption reaction vessel and culturing by using a complete culture medium, wherein the complete culture medium is firstly used for culturing at 37 DEG CShake culturing for 20min at a table rotation speed of 40r/min, stopping shaking, standing, and continuing culturing for 3 days under the following conditions: the temperature is 37.0 +/-0.5 ℃ and CO2The concentration is 5.0 +/-0.2%, and the humidity is saturated humidity.
4. The method of culturing the mesenchymal stem cell of any one of claims 1-3, wherein the mesenchymal stem cell comprises one of adipose mesenchymal stem cell, umbilical cord mesenchymal stem cell and endometrial mesenchymal stem cell.
5. The method of claim 4, wherein the mesenchymal stem cell is adipose mesenchymal stem cell, and the adipose mesenchymal stem cell suspension is prepared by the following steps:
(1) taking fat cells;
(2) placing the fat cells in a test tube, adding digestive juice with the volume same as that of the fat tissue into the test tube, wherein the digestive juice comprises trypsin-EDTA and 0.1% type I collagenase in a volume ratio of 1:1, sealing the test tube, placing the test tube in a constant-temperature shaking table at 37 ℃, and performing shock digestion at a speed of 180-;
(3) sucking the lower layer liquid in the centrifuge tube, transferring into a new centrifuge tube filled with complete culture medium to terminate digestion, sealing the centrifuge tube, centrifuging, and separating into 2 layers;
(4) sucking the supernatant in the new centrifugal tube by using a suction tube, removing the supernatant, adding a complete culture medium, and slightly blowing and beating to prepare cell suspension;
(5) the cell suspension was adjusted to 0.5X 106Inoculating to culture flask at density of one ml, placing at 37 deg.C and 5% CO2Culturing in an incubator; carrying out first liquid change after 12-24 hours, changing liquid every 3 days, and carrying out passage after cells grow to be fused;
(6) sucking out the old culture medium in the culture bottle, adding PBS for washing for 2-3 times, and adding digestive juice for digestion for 3-5min, wherein the digestive juice is trypsin-EDTA; and (4) gently blowing and beating the cells to separate the cells from the bottom of the bottle to obtain single cell suspension.
6. The method for culturing mesenchymal stem cells on an acellular allogeneic dermal scaffold according to claim 5, wherein the adipose tissue in the step (1) is adipose tissue directly obtained by liposuction.
7. The method for culturing mesenchymal stem cells on an acellular allogeneic dermal scaffold according to claim 4, wherein the method for obtaining the adipocytes in the step (1) comprises: squeezing adipose tissue into a centrifuge tube, sucking PBS buffer solution, adding into the centrifuge tube with tissue, using 12-16ml PBS buffer solution per gram of adipose tissue, blowing with a pipette for 10-20 times, standing for 0.5-1.5min, sucking out the liquid in the lower layer of tissue with the pipette, and repeating the above steps until the liquid is transparent and has no blood cells.
8. The method of claim 1, wherein the acellular allogeneic dermal scaffold is cut into square small pieces with a side length of 2cm and washed three times with physiological saline before being placed in the ultra-low adsorption reaction dish.
9. An artificial skin prepared by the method of culturing the mesenchymal stem cells according to any one of claims 1 to 8 on an acellular allogeneic dermal scaffold.
10. Use of the artificial skin according to claim 7 in the medical field of biomimetic materials.
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