CN112831465A - Culture method of autologous adipose-derived stem cells for treating central nervous system injury - Google Patents
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Abstract
The invention discloses a method for culturing autologous adipose-derived stem cells for treating central nervous system injury, which comprises the following steps of firstly, fat collection, and then, collecting the adipose of a donor in a sterile place to obtain adipose tissues; step two, obtaining and separating autologous adipose-derived stem cells: digesting the adipose tissues and the taken adipose tissues by using 0.2% collagenase phosphate buffer solution with the same volume, and cleaning; filtering, removing undigested tissue, centrifuging for 5min, and discarding supernatant to obtain stem cells; step three, stem cell culture: the stem cells obtained in the second step are treated in a ratio of 1-2X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in 5% incubator, centrifuging after culturing, discarding supernatant, adding 30mL dry cell culture solution containing 15% fetal calf serum by volume fraction into centrifuge tube, and blowing to clean and mix cells to obtain final productBody adipose-derived stem cells.
Description
Technical Field
The invention belongs to the technical field of stem cell culture, and particularly relates to a culture method of autologous adipose-derived stem cells for treating central nervous system injury.
Background
The central nervous system, which contains the brain and spinal cord, is the center of all sensory perceptions and motor outputs. The ability of the central nervous system to repair and regenerate is very limited because damaged/axon-incised neurons and most transected central nerve axons are non-regenerable. The central nervous system injury comprises traumatic brain injury and spinal cord injury, is the main reason of clinical traumatic death and disability, and with the continuous and deep research on stem cells, more and more evidences show that the neural stem cells can improve the spinal cord injury repair effect to a certain extent. The mechanism is mainly that the neural stem cells paracrine a large amount of nerve regeneration factors, such as BDNF, NT-3, GDNF, NGF and the like, so that the transplantation of the neural stem cells mainly acts in a paracrine mode. Moreover, the concentration of the secreted factors gradually decreases with the death of the transplanted cells in vivo. The method comprises the steps of separating and concentrating a plurality of regeneration factors secreted into a culture medium by stem cells cultured in vitro, and then compounding the regeneration factors on a biological material for repairing the central nervous system.
Disclosure of Invention
The present invention provides a method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury.
The technical problems to be solved by the invention are as follows:
the central nervous system injury comprises traumatic brain injury and spinal cord injury, is the main reason of clinical traumatic death and disability, and with continuous and deep research on stem cells, more and more evidences show that the neural stem cells can improve the spinal cord injury repair effect to a certain extent.
The purpose of the invention can be realized by the following technical scheme:
a method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, then mincing, washing with a D-Hanks buffer solution containing gentamicin with the volume fraction of 0.1-1% after mincing, and removing residual blood to obtain fat tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting the adipose tissues and the taken adipose tissues by using a 0.2% collagenase phosphate buffer solution with the same volume, uniformly shaking and digesting the adipose tissues and the taken adipose tissues for 50 to 70 minutes at the temperature of 37 ℃, and then placing the adipose tissues and the taken adipose tissues in a centrifuge for centrifuging the adipose tissues for 6 to 10 minutes at the rotating speed of 1800 and 2000 r/min; repeatedly beating the bottom layer cells with D-Hanks balanced salt solution with pH value of 7.2-7.4, and cleaning; filtering the cleaned liquid by a 100-mesh screen, removing undigested tissues, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell culture: the stem cells obtained in the second step are treated in a ratio of 1-2X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 4 to 5 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 3 to 4 days, and adding pancreatin-EDTA with the volume fraction of 0.25 percent into a culture bottle for digestion when the cells grow to reach 80 percent; placing the culture bottles in an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently patting the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum by volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotation speed of 900 plus material 1000r/min, removing the supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum by volume fraction into the centrifuge tube, blowing, cleaning and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
Further, a stem cell culture solution is prepared by the following steps:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
Further, the sterilization solution is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
The invention has the beneficial effects that:
the low-sugar DMEM culture medium can effectively provide required nutrient components for the amplification of mesenchymal stem cells from adipose tissues, and on the basis of the low-sugar DMEM culture medium, by adding functional components of fetal bovine serum and gentamicin, the two functional components can play a synergistic and synergistic role, so that the efficient amplification of the stem cells from the adipose tissues is realized, the proliferation of neural stem cells can be promoted by matching non-essential amino acid and beta-mercaptoethanol, the addition of a bacteria removing solution ensures that the cultured stem cells are not subjected to bacterial pollution under the condition of no differentiation.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, mincing, washing with a D-Hanks buffer solution containing gentamicin with a volume fraction of 0.1% after mincing, and removing residual blood to obtain adipose tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting adipose tissues and 0.2% collagenase phosphate buffer solution with the same volume of the taken adipose tissues, uniformly shaking and digesting for 50min at the temperature of 37 ℃, and then placing the digested adipose tissues in a centrifuge to centrifuge for 6min at the rotating speed of 1800 r/min; repeatedly beating the bottom layer cells by using D-Hanks balanced salt solution with the pH value of 7.2, and cleaning; filtering the cleaned liquid by a 100-mesh screen, removing undigested tissues, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell culture: the stem cells obtained in the second step are treated at a ratio of 1X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 4 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 3 days, and adding pancreatin-EDTA with the volume fraction of 0.25 percent into a culture bottle for digestion when the cell growth reaches 80 percent; putting the culture bottles into an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently beating the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotation speed of 900r/min, removing supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into the centrifuge tube, blowing and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
Wherein, the stem cell culture solution is prepared by the following steps:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
The bacteria removing liquid is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
Example 2
A method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, mincing, washing with a D-Hanks buffer solution containing gentamicin with a volume fraction of 0.6% after mincing, and removing residual blood to obtain adipose tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting adipose tissues and 0.2% collagenase phosphate buffer solution with the same volume of the taken adipose tissues, uniformly shaking and digesting for 60min at the temperature of 37 ℃, and then placing the digested adipose tissues in a centrifuge to centrifuge for 8min at the rotating speed of 1900 r/min; repeatedly beating the bottom layer cells by using D-Hanks balanced salt solution with the pH value of 7.3, and cleaning; filtering the cleaned liquid by a 100-mesh screen, removing undigested tissues, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell culture: the stem cells obtained in the second step are treated at a ratio of 1X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 4 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 3 days, and adding pancreatin-EDTA with the volume fraction of 0.25 percent into a culture bottle for digestion when the cell growth reaches 80 percent; putting the culture bottles into an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently beating the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotation speed of 950r/min, removing supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into the centrifuge tube, blowing and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
Wherein, the stem cell culture solution is prepared by the following steps:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
The bacteria removing liquid is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
Example 3
A method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, mincing, washing with D-Hanks buffer solution containing gentamicin with the volume fraction of 1%, and removing residual blood to obtain adipose tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting adipose tissues and 0.2% collagenase phosphate buffer solution with the same volume of the taken adipose tissues, uniformly shaking and digesting for 70min at the temperature of 37 ℃, and then placing the digested adipose tissues in a centrifuge to centrifuge for 10min at the rotating speed of 2000 r/min; repeatedly beating the bottom layer cells by using D-Hanks balanced salt solution with the pH value of 7.4, and cleaning; filtering the cleaned liquid by a 100-mesh screen, removing undigested tissues, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell culture: the stem cells obtained in the second step are mixed according to the ratio of 2X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 5 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 4 days,when the cell growth reaches 80% fusion, adding pancreatin-EDTA with the volume fraction of 0.25% into a culture bottle for digestion; putting the culture bottles into an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently beating the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotating speed of 1000r/min, removing supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into the centrifuge tube, blowing and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
Wherein, the stem cell culture solution is prepared by the following steps:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
The bacteria removing liquid is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
Example 4
A method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, mincing, washing with a D-Hanks buffer solution containing gentamicin with a volume fraction of 0.1% after mincing, and removing residual blood to obtain adipose tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting adipose tissues and 0.2% collagenase phosphate buffer solution with the same volume of the taken adipose tissues, uniformly shaking and digesting for 70min at the temperature of 37 ℃, and then placing the digested adipose tissues in a centrifuge to centrifuge for 10min at the rotating speed of 2000 r/min; repeatedly beating the bottom layer cells by using D-Hanks balanced salt solution with the pH value of 7.3, and cleaning; filtering the cleaned liquid by a 100-mesh screen, removing undigested tissues, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell culture: the stem cells obtained in the second step are mixed according to the ratio of 2X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 5 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 4 days, and adding pancreatin-EDTA with the volume fraction of 0.25 percent into a culture bottle for digestion when the cell growth reaches 80 percent; putting the culture bottles into an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently beating the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotating speed of 1000r/min, removing supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum in volume fraction into the centrifuge tube, blowing and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
Wherein, the stem cell culture solution is prepared by the following steps:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
The bacteria removing liquid is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
The foregoing is illustrative and explanatory only and is not intended to be exhaustive or to limit the invention to the precise embodiments described, and various modifications, additions, and substitutions may be made by those skilled in the art without departing from the scope of the invention or exceeding the scope of the claims.
Claims (3)
1. A method for culturing autologous adipose-derived stem cells for the treatment of central nervous system injury, comprising the steps of:
firstly, collecting fat, collecting the fat of a donor in a sterile place, treating the obtained fat, removing blood vessels and fiber parts, then mincing, washing with a D-Hanks buffer solution containing gentamicin with the volume fraction of 0.1-1% after mincing, and removing residual blood to obtain fat tissue;
step two, obtaining and separating autologous adipose-derived stem cells: digesting the adipose tissues and the taken adipose tissues by using a 0.2% collagenase phosphate buffer solution with the same volume, uniformly shaking and digesting the adipose tissues and the taken adipose tissues for 50 to 70 minutes at the temperature of 37 ℃, and then placing the adipose tissues and the taken adipose tissues in a centrifuge for centrifuging the adipose tissues for 6 to 10 minutes at the rotating speed of 1800 and 2000 r/min; repeatedly beating the bottom layer cells with D-Hanks balanced salt solution with pH value of 7.2-7.4, and cleaning; filtering the cleaned liquid by a 100-mesh screen, centrifuging the filtrate at 1000r/min for 5min, and removing the supernatant to obtain stem cells;
step three, stem cell cultureCulturing: the stem cells obtained in the second step are treated in a ratio of 1-2X 104/cm2Inoculating into culture bottle, adding 10mL stem cell culture solution containing 15% volume fraction fetal calf serum, placing at 37 deg.C and CO2Culturing in an incubator with the content of 5 percent, replacing a new stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent after 4 to 5 days, discarding non-adherent cells, replacing the stem cell culture solution containing fetal calf serum with the volume fraction of 15 percent once every 3 to 4 days, and adding pancreatin-EDTA with the volume fraction of 0.25 percent into a culture bottle for digestion when the cells grow to reach 80 percent; placing the culture bottles in an incubator at 37 ℃ for 5min, observing cells under an inverted microscope, gently patting the culture bottles with palms to ensure that the cells and tissue blocks are completely digested, adding 10mL of stem cell culture solution containing 15% fetal calf serum by volume fraction into each culture bottle, repeatedly blowing, cleaning the cells in the culture bottles, transferring the cleaned cell suspension into a 50mL centrifuge tube, centrifuging for 5min at the rotation speed of 900 plus material 1000r/min, removing the supernatant after centrifugation is finished, adding 30mL of stem cell culture solution containing 15% fetal calf serum by volume fraction into the centrifuge tube, blowing, cleaning and uniformly mixing the cells to obtain the autologous adipose-derived stem cells.
2. The method of claim 1, wherein the stem cell culture fluid is prepared by the steps of:
1% of L-glutamine, 1% of nonessential amino acid, 1% of sodium pyruvate, 0.01% of beta-mercaptoethanol, 0.001% of LLIF, 0.0001% of bFGF, 10% of FBS and 0.001% of bacteria removing liquid are subjected to constant volume to 100mL by using low-sugar DMEM, filtered by using a 0.22 mu L filter and stored at 0 ℃ to obtain a stem cell culture solution, wherein the percentage of the stem cell culture solution is the volume percentage of the prepared stem cell culture solution.
3. The culture method of the autologous adipose-derived stem cells for treating the central nervous system injury is characterized in that a sterilization solution is prepared by the following steps:
200U of streptomycin, 100 ug of kanamycin, 15 ug of nystatin and 5 ug of amphotericin B were mixed, and the volume was adjusted to 100mL with D-PBS to obtain a sterilized solution.
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| CN202110036915.8A CN112831465A (en) | 2021-01-12 | 2021-01-12 | Culture method of autologous adipose-derived stem cells for treating central nervous system injury |
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| CN202110036915.8A CN112831465A (en) | 2021-01-12 | 2021-01-12 | Culture method of autologous adipose-derived stem cells for treating central nervous system injury |
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