CN112773759A - Preparation method of paracrine factor inclusion body with skin cell rejuvenation effect - Google Patents
Preparation method of paracrine factor inclusion body with skin cell rejuvenation effect Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
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- A61K8/00—Cosmetics or similar toiletry preparations
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- A61K8/11—Encapsulated compositions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
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Abstract
The invention discloses a preparation method of a paracrine factor inclusion body with skin cell rejuvenation effect, which comprises the following steps: step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation; step b: placing the umbilical cord in a culture dish, fully washing the umbilical cord with physiological saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small sections with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly for 3 times with the physiological saline, and shearing the Wharton's jelly into tissue blocks with the size of 1-3 cubic millimeters. The mesenchymal stem cell paracrine factor is encapsulated by the microcapsule, can be stored at normal temperature, has stable property, and can improve the increase of collagen secretion of the fibroblast, improve the activity of division and prolong the service life of the fibroblast by co-culturing the microcapsule inclusion body and the fibroblast.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method of a paracrine factor inclusion with skin cell rejuvenation effect.
Background
The mesenchymal stem cells are pluripotent stem cells derived from mesoderm and ectoderm in early development, and the MSC has the functions of immunoregulation, promotion of homing of hematopoietic stem cells, repair of damaged or diseased tissues and organs, multidirectional differentiation potential and the like, so that the mesenchymal stem cells have wide application prospects.
With the continuous and deep research on the mesenchymal stem cells, the action mechanism of the mesenchymal stem cells is clearer and clearer, and the mesenchymal stem cells mainly have 3 aspects: 1. the paracrine action of the mesenchymal stem cells, 2, the mesenchymal stem cells replace or repair dead or damaged tissue cells, 3, the cells are in direct contact and regulate and control the cells, more and more researches show that the paracrine action of the mesenchymal stem cells plays a main role in playing the treatment effect, the umbilical cord mesenchymal stem cells can secrete various cytokines such as epidermal growth factors, vascular endothelial growth factors, fibroblast growth factors, hepatocyte growth factors, stromal cell derived factors-1, tumor necrosis factors, nerve growth factors, basic fibroblast growth factors, interleukin 6 and the like in the culture process, the factors secreted by the mesenchymal stem cells have the functions of improving the cell growth microenvironment, regulating the organism immunity, promoting the blood vessel regeneration, promoting the cell proliferation, inhibiting the cell apoptosis and the like, and have good repairing and repairing effects on damaged skin, sensitive skin and reconstructed skin, Has effects of caring skin, promoting collagen synthesis, and delaying aging.
The paracrine cell factor is purified and concentrated and then added into the beauty cosmetics, which is a new trend for the development of the cosmetic industry, the paracrine cell factor cosmetics are particularly suitable, but the paracrine cell factor cosmetics on the market are generally produced by directly mixing stem cell culture supernatant into a cosmetic matrix, and have low paracrine cell factor content, poor uniformity, poor stability and general beauty, skin care and anti-aging effects.
In the prior art, a common culture bottle is used for culturing mesenchymal stem cells, collecting and merging multi-generation cell culture supernatant, and filtering the supernatant to obtain the paracrine cell factor, the content of the paracrine cell factor of the mesenchymal stem cells obtained by the method is low, the method needs to collect multi-generation cell culture medium, the efficiency is low, the uniformity of products is difficult to guarantee due to the fact that the paracrine cell factor is prepared by the multi-generation collection method, in addition, the paracrine cell factor prepared by the method is liquid, is easy to degrade at normal temperature, needs to be stored at low temperature, and increases the storage and transportation difficulty.
Disclosure of Invention
The invention aims to provide a preparation method of a paracrine factor inclusion body with the skin cell rejuvenation effect, has the advantage of convenient use of the paracrine factor inclusion body, and solves the problem of inconvenient use of the paracrine factor.
In order to achieve the purpose, the invention provides the following technical scheme: the preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps:
step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation;
step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord by using normal saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small sections with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly for 3 times by using normal saline, shearing the Wharton's jelly into tissue blocks with the size of 1-3 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with the temperature of 37 ℃ and 5% CO2 for adherence for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture box in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage;
step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 3-5 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 70% -80% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h;
step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 1.5-2h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing in a 4-DEG refrigerator, transferring all the primarily concentrated extract into an ultrafiltration tube, further concentrating for 1.5-2h, discarding the liquid below the ultrafiltration tube, adding saline to repeat ultrafiltration and centrifugation washing once, collecting the concentrated liquid to obtain a concentrated stock solution of the paracrine factor of the mesenchymal stem cells, and wrapping by microcapsules to obtain a microcapsule inclusion body;
step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Preferably, the storage and transportation solution in the step a is 40ml of alpha-MEM, and 100U/ml of gentamicin, 50mg/ml of penicillin and 50mg/ml of amphotericin B5ug/ml are added.
Preferably, the serum-free complete medium in the step b is: serum replacement at a volume concentration of 2% and glutamine dipeptide at a volume concentration of 1%, the remainder being LONZA serum-free medium.
Preferably, the umbilical cord of the newborn in healthy term is taken in the step a, and the umbilical cord is 20cm close to the end of the infant.
Compared with the prior art, the invention has the following beneficial effects: the mesenchymal stem cell paracrine factor is encapsulated by the microcapsule, can be stored at normal temperature, has stable property, and can improve the increase of collagen secretion of the fibroblast, improve the activity of division and prolong the service life of the fibroblast by co-culturing the microcapsule inclusion body and the fibroblast.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps:
step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation;
step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord by using normal saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small sections with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly for 3 times by using normal saline, shearing the Wharton's jelly into tissue blocks with the size of 1-3 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with the temperature of 37 ℃ and 5% CO2 for adherence for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture box in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage;
step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 3-5 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 70% -80% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h;
step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 1.5-2h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing in a 4-DEG refrigerator, transferring all the primarily concentrated extract into an ultrafiltration tube, further concentrating for 1.5-2h, discarding the liquid below the ultrafiltration tube, adding saline to repeat ultrafiltration and centrifugation washing once, collecting the concentrated liquid to obtain a concentrated stock solution of the paracrine factor of the mesenchymal stem cells, and wrapping by microcapsules to obtain a microcapsule inclusion body;
step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Example 1
The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps: step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation; step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord with physiological saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small segments with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly with physiological saline for 3 times, shearing the Wharton's jelly into tissue blocks with the size of 1 cubic millimeter, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with 37 ℃, adhering the tissue blocks to the wall for 2 hours in the culture box with 5% CO2, adding 15ml of serum-free complete culture medium, placing the culture box in the culture box for culture, keeping the culture box absolutely, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage; step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 5 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 70% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h; step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 1.5h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing the concentrated liquid in a 4-DEG refrigerator, transferring all the preliminarily concentrated extract into the ultrafiltration tube, further concentrating for 1.5h, discarding the liquid below the ultrafiltration tube, adding saline, repeatedly performing ultrafiltration and centrifugation washing for one time, collecting the concentrated liquid to obtain a mesenchymal stem cell paracrine factor concentrated stock solution, and wrapping the umbilical cord microcapsule inclusion by virtue of microcapsules to obtain an umbilical cord microcapsule inclusion body; step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Example 2
In example 1, the following steps were added:
in the step a, the storage and transportation liquid is 40ml of alpha-MEM, and 100U/ml of gentamicin, 50mg/ml of penicillin and 5ug/m of amphotericin are added.
The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps: step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation; step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord with physiological saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small segments with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly with physiological saline for 3 times, shearing the Wharton's jelly into tissue blocks with the size of 2 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with 37 ℃, 5% CO2 for adhering to the wall for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture bottle in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage; step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 3 multiplied by 104/ml, marking, placing the cells in a 37 ℃ and 5% CO2 incubator for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 to continue culture when the cells subjected to amplification culture grow to 75% of the cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 to continue culture, and collecting cell supernatant after 72 h; step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 2h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing the concentrated liquid in a 4-DEG refrigerator, transferring all the primarily concentrated extract into an ultrafiltration tube, further concentrating for 1.5h, discarding the liquid below the ultrafiltration tube, adding saline, repeatedly performing ultrafiltration and centrifugation washing once, collecting the concentrated liquid to obtain a concentrated stock solution of the umbilical mesenchymal stem cell paracrine secretion factor, and wrapping by microcapsules to obtain a microcapsule inclusion body; step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Example 3
In example 2, the following steps were added:
the serum-free complete culture medium in the step b is as follows: serum replacement at a volume concentration of 2% and glutamine dipeptide at a volume concentration of 1%, the remainder being LONZA serum-free medium.
The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps: step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation; step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord with physiological saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small segments with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly with physiological saline for 3 times, shearing the Wharton's jelly into tissue blocks with the size of 3 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with 37 ℃, 5% CO2 for adhering to the wall for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture bottle in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage; step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 4 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 80% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h; step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 2h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing the concentrated liquid in a 4-DEG refrigerator, transferring all the primarily concentrated extract into an ultrafiltration tube, further concentrating for 2h, discarding the liquid below the ultrafiltration tube, adding saline, repeatedly performing ultrafiltration and centrifugation for one time, collecting the concentrated liquid to obtain a concentrated raw solution of the paracrine factor of the mesenchymal stem cells, and wrapping the concentrated raw solution by microcapsules to obtain a microcapsule inclusion body; step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Example 4
In example 3, the following steps were added:
taking the umbilical cord of a healthy and full-term newborn in the step a, wherein the umbilical cord is 20cm close to the end of the infant.
The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect comprises the following steps: step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation; step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord with physiological saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small segments with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly with physiological saline for 3 times, shearing the Wharton's jelly into tissue blocks with the size of 2 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with 37 ℃, 5% CO2 for adhering to the wall for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture bottle in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage; step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 5 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 70% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h; step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 1.5h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing the concentrated liquid in a 4-DEG refrigerator, transferring all the preliminarily concentrated extract into the ultrafiltration tube, further concentrating for 1.5h, discarding the liquid below the ultrafiltration tube, adding saline, repeatedly performing ultrafiltration and centrifugation washing for one time, collecting the concentrated liquid to obtain a mesenchymal stem cell paracrine factor concentrated stock solution, and wrapping the umbilical cord microcapsule inclusion by virtue of microcapsules to obtain an umbilical cord microcapsule inclusion body; step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. The preparation method of the paracrine factor inclusion body with the skin cell rejuvenation effect is characterized in that: the method comprises the following steps:
step a: placing the umbilical cord in a storage and transportation bottle containing storage and transportation liquid, and preparing the umbilical cord by separation;
step b: placing an umbilical cord in a culture dish, fully washing the umbilical cord by using normal saline, shearing off the length of 1cm from each end of the umbilical cord, repeatedly washing umbilical cord tissues until no obvious blood clots exist, shearing the umbilical cord into small sections with the length of 2cm, washing for 3 times, longitudinally splitting the umbilical cord, removing blood vessels, tearing Wharton's jelly, fully washing the Wharton's jelly for 3 times by using normal saline, shearing the Wharton's jelly into tissue blocks with the size of 1-3 cubic millimeters, inoculating the tissue blocks into a culture bottle, placing the culture bottle in a culture box with the temperature of 37 ℃ and 5% CO2 for adherence for 2 hours, adding 15ml of serum-free complete culture medium, placing the culture box in the culture box for culture, keeping the culture box absolutely static, observing under a microscope, wherein fibroblasts climb out around the tissue block, the number of the 12d cells is large, removing the tissue block, digesting with trypsin to transfer adherent cells into a new culture bottle for culture, and then carrying out passage;
step c: detecting the cultured cells, transferring the qualified cells to a cell factory according to the final concentration of 3-5 multiplied by 104/ml, marking, placing the cells in an incubator at 37 ℃ and 5% CO2 for amplification culture, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture when the cells subjected to amplification culture grow to 70% -80% of cell fusion degree, adjusting the parameters to 37 ℃, 5% CO2 and 15% O2 after 24h, collecting half amount of supernatant after 48h, supplementing an equivalent amount of fresh serum-free culture medium into the cell factory, adjusting the parameters to 37 ℃, 5% CO2 and 5% O2 for continuous culture, and collecting cell supernatant after 72 h;
step d: putting the collected mesenchymal stem cell culture supernatant into a centrifuge tube, centrifuging for 8min at 4 ℃ and 4000g, taking the supernatant, removing cell sediments, filtering the supernatant through a hollow fiber filter membrane, collecting filtrate, transferring the filtrate without sediments into a 5KD ultrafiltration concentration centrifuge tube by using a pipette, centrifuging for 1.5-2h at 4 ℃ and 4000g, collecting the concentrated liquid into a new centrifuge tube, storing in a 4-DEG refrigerator, transferring all the primarily concentrated extract into an ultrafiltration tube, further concentrating for 1.5-2h, discarding the liquid below the ultrafiltration tube, adding saline to repeat ultrafiltration and centrifugation washing once, collecting the concentrated liquid to obtain a concentrated stock solution of the paracrine factor of the mesenchymal stem cells, and wrapping by microcapsules to obtain a microcapsule inclusion body;
step e: the microcapsule inclusion is re-suspended in culture medium containing fibroblast in the culture box and inoculated in a culture flask, and observation under a microscope shows that the fibroblast secretes increased collagen, the division is active and the service life is prolonged.
2. The method for producing a paracrine factor inclusion body having skin cell rejuvenation effect according to claim 1, wherein: the storage and transportation liquid in the step a is 40ml of alpha-MEM, and 100U/ml of gentamicin, 50mg/ml of penicillin and 5ug/ml of amphotericin are added.
3. The method for producing a paracrine factor inclusion body having skin cell rejuvenation effect according to claim 1, wherein: the serum-free complete culture medium in the step b is as follows: serum replacement at a volume concentration of 2% and glutamine dipeptide at a volume concentration of 1%, the remainder being LONZA serum-free medium.
4. The method for producing a paracrine factor inclusion body having skin cell rejuvenation effect according to claim 1, wherein: in the step a, the umbilical cord of a healthy and full-term newborn is taken, and the umbilical cord is 20cm close to the end of the infant.
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| CN113425619A (en) * | 2021-05-18 | 2021-09-24 | 铜仁市泛特尔生物技术有限公司 | Purification preparation method and application of mesenchymal stem cell secretory factor |
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| 国家食品药品监督管理总局: "化妆品禁用组分", 《化妆品安全技术规范》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113425619A (en) * | 2021-05-18 | 2021-09-24 | 铜仁市泛特尔生物技术有限公司 | Purification preparation method and application of mesenchymal stem cell secretory factor |
| CN113425619B (en) * | 2021-05-18 | 2023-04-28 | 铜仁市泛特尔生物技术有限公司 | Purification preparation method and application of mesenchymal stem cell secretion factor |
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Application publication date: 20210511 |