CN114306749A - Preparation and application of living cell-loaded placenta acellular matrix gel - Google Patents
Preparation and application of living cell-loaded placenta acellular matrix gel Download PDFInfo
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Abstract
The invention discloses a preparation method of placenta acellular matrix gel loaded with living cells, which comprises the following steps: pretreating placenta tissues, performing virus inactivation treatment on the placenta tissues, removing virus inactivation solution residues, performing three times of circulating freeze thawing treatment on the tissues, performing decellularization treatment, removing the cell removal solution residues, drying and performing ball milling on the tissues, and performing irradiation sterilization; after the powder is sterilized, adding normal saline to dissolve the powder, adding the extracted living cells, uniformly mixing, placing the mixture into different molds, and placing the molds into an incubator at 37 ℃ for 10-20min to form gel. The invention adopts the preparation and the application of the placenta acellular matrix gel loaded with the living cells, the prepared gel can load various cells and support the growth of the cells, the gelling time is short, and the vitality and the death rate of the cells can be improved.
Description
Technical Field
The invention relates to the technical field of medical science and beauty, in particular to preparation and application of a living cell-loaded placenta acellular matrix gel.
Background
Acellular matrix (dmem) has been widely used as a scaffold material for regenerative medicine due to its high biomimetic property and excellent biocompatibility. The hydrogel (hydrogel) is a functional polymer material with high water content and controllable fluidity, and is very suitable for partial clinical minimally invasive surgery. With the rapid development of hydrogel theory and technology, dECM hydrogels are becoming the research hot spot in the field of regenerative medicine.
At present, pig placenta is commonly used as a raw material of a acellular matrix, and is transported in a low-temperature cold chain transportation mode after virus inactivation is carried out by using a peracetic acid solution at present. The preparation of acellular matrix includes physical, chemical and enzymatic methods. The hydrogel takes a three-dimensional fiber network material with good biocompatibility as a framework, namely, the acellular matrix hydrogel contains the three-dimensional fiber network material with good biocompatibility and is directly compounded with the acellular matrix gel, and the aim of enhancing the acellular matrix hydrogel can be achieved without changing the structure of the acellular matrix gel.
CN201610395766.3 bionic tissue based on bladder acellular matrix-cell-three-dimensional fiber network and preparation thereof, the disclosed bionic tissue at least comprises composite bladder acellular matrix hydrogel and epithelial cells; the composite bladder acellular matrix hydrogel is a bladder acellular matrix hydrogel taking a three-dimensional fiber network material with good biocompatibility as a framework.
In the prior art, the cell removing method adopts glutaraldehyde for cell removal, the glutaraldehyde can denature the product, and the glutaraldehyde is difficult to remove. The DNase working solution specifically removes DNA, the cost of the enzyme is high, and other immunogenic substances are introduced. After decellularization, most products are insoluble collagen products, cannot be directly used as carriers to load cells, and can form gel only by compounding other materials, so that the cost is increased.
Disclosure of Invention
The invention aims to provide preparation and application of a living cell-loaded placenta acellular matrix gel, the prepared gel can load various cells and support the growth of the cells, the gelling time is short, and the vitality and the death rate of the cells can be improved.
In order to realize the aim, the invention provides a preparation method of a placenta acellular matrix gel loaded with living cells, which comprises the following steps:
s1, pretreatment of tissues: selecting fresh placenta delivered within 6h, cleaning blood stain with clear water, soaking in sterile storage solution containing peroxyacetic acid and ethanol for 0.5-4h, and storing placenta tissue at-8 deg.C for a long time;
s2, inactivating viruses of the placenta tissues: adding 20L virus inactivating solution into per kilogram of placenta tissue, and treating for 5-10 hr;
s3, removing the virus inactivation solution residues: centrifuging the inactivated placenta, removing supernatant, adding purified water with the same amount for cleaning, centrifuging again, removing supernatant, and repeating for 2-3 times;
s4, carrying out three times of circulating freeze-thaw treatment on the tissue obtained in the step S3;
s5, cell removal treatment: treating the tissue treated in the step S4 with protease solution, nonionic surfactant solution and ionic surfactant solution for 90-120min, wherein stirring is continued;
s6, removing the cell removing solution residue: washing the tissue treated in the step S5 with purified water for 15-30min, centrifuging, removing supernatant, and repeating for 4-6 times;
s7, drying and ball-milling the tissue processed in the step S6, and then performing irradiation sterilization;
s8, adding normal saline into the sterilized powder obtained in the step S7 to dissolve the powder, adding the extracted living cells, uniformly mixing, placing the mixture into different molds, and placing the molds into an incubator at 37 ℃ for 10-20min to form gel.
Preferably, in step S1, the placenta is transported by cold chain transport at a temperature below 0 ℃.
Preferably, in step S2, the virus inactivation solution is prepared from 1% -3% peracetic acid, absolute ethanol and purified water in a volume ratio of 3: 5: 92 to mix.
Preferably, in step S4, the three-cycle freeze-thaw treatment process includes: the freezing temperature is-80 ℃ to-40 ℃, and the freezing time is not less than 10 hours; the melting temperature is 20-28 ℃, after each melting, the supernatant is centrifuged and discarded, and the same amount of purified water is added again for freezing.
Preferably, the protease solution in step S5 is 0.20% -0.25% trypsin,
the non-ionic surfactant solution is 0.10-0.15% triton solution,
the ionic surfactant solution is 0.10-0.15% sodium dodecyl sulfate.
Preferably, in step S7, the drying and ball milling process includes:
first freeze drying: placing the tissue in a freeze-drying tray, pre-freezing for 6-8h at-60- -48 ℃, and carrying out gradient freezing and vacuum pumping, wherein the temperature of each gradient is increased by 10 ℃ to obtain acellular freeze-dried powder;
first freezing ball milling: ball milling the dried acellular placenta matrix at low temperature, and passing the ball-milled tissue through a 30-mesh sieve;
and (3) second freeze drying: digesting the cell-free freeze-dried powder obtained after ball milling with an acid enzyme solution, adjusting the pH value to 6.0-7.0 with an alkaline solution, pouring into a tray, pre-freezing for 6-8 hours at-60 to-48 ℃, performing gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain cell-free porous sponge;
and (3) performing second freezing ball milling: the obtained porous sponge is subjected to freezing ball milling at the temperature of-20 ℃ to-50 ℃, and the ball-milled tissue passes through a 50-mesh sieve to obtain particle powder with uniform particle size.
Application of placenta acellular matrix gel loaded with living cells in medical beauty, face-lifting and female health is provided.
Therefore, the preparation and application of the living cell-loaded placenta acellular matrix gel have the following specific effects:
(1) the invention adopts a virus inactivation technology, the time of the virus inactivation technology is short, and after verification, the requirements of the animal-derived medical instrument registration technology review guide principle (revised version in 2017) are met, and the safety of the product can be ensured. The placenta is biological waste, does not relate to ethical problem, and has rich material sources. Moreover, the pig placenta has sufficient yield and low price.
(2) The gel prepared by the invention is only an acellular matrix and can meet the requirements without compounding other substances. The gel can support a variety of cells and support the growth of cells, such as: C2C12 myoblasts, SH-SY5Y neuroblastoma cells, BMSCs bone marrow mesenchymal stem cells, PMSCs placenta mesenchymal stem cells and other stem cells, and the survival of the cells is ensured.
(3) The gel is preserved in the form of freeze-dried powder, and is dissolved in water when cells need to be loaded, so that the preservation time can be prolonged. In the absence of nutrients, the gelling time is short, and the vitality and death rate of cells can be improved.
(4) In the cell removing process, the used reagent and the method are simple, and not only can remove cell nucleuses, but also can retain extracellular matrix components and results.
(5) The freezing ball milling process ensures that the ball milling process is maintained at the low temperature of-20 ℃ to-50 ℃, avoids protein denaturation caused by overhigh temperature of products due to friction, can randomly adjust the proportion of ball milling balls to adapt to different types of substances, and has the ball milling yield of over 200g per day. The production line period is 15 days, the production capacity of each batch can reach 150g, and the yield meets the clinical requirement.
The technical solution of the present invention is further described in detail by the accompanying drawings and embodiments.
Drawings
FIG. 1 is a process flow diagram of the preparation and application of a viable cell loaded placental acellular matrix gel of the present invention;
FIG. 2 is a bar graph of the effect of different indicators after the subject uses the gel;
FIG. 3 is a pathological section of the uterine cavity adhesion group and the gel injection group.
Detailed Description
The technical solution of the present invention is further illustrated by the accompanying drawings and examples.
Unless defined otherwise, technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. The use of "first," "second," and similar terms in the present application do not denote any order, quantity, or importance, but rather the terms are used to distinguish one element from another. The word "comprising" or "comprises", and the like, means that the element or item listed before the word covers the element or item listed after the word and its equivalents, but does not exclude other elements or items. The terms "connected" or "coupled" and the like are not restricted to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "upper", "lower", "left", "right", and the like are used merely to indicate relative positional relationships, and when the absolute position of the object being described is changed, the relative positional relationships may also be changed accordingly.
Example one
A preparation method of placenta acellular matrix gel loaded with living cells comprises the following steps:
(1) selecting materials for placenta tissues: fresh placenta delivered within 6 hours.
(2) Collecting and transporting placenta tissues: collecting fresh placenta, cleaning with clear water to remove blood stain, soaking in sterile storage solution containing peroxyacetic acid and ethanol for 1 hr, and storing at-8 deg.C for a long period. The placenta is transported in a low-temperature cold chain (less than 0 ℃).
(3) And (3) inactivating the viruses of the placenta tissues: the virus inactivation solution is prepared by mixing peroxyacetic acid, absolute ethyl alcohol and purified water according to equal volume ratio. 1kg of placental tissue was treated with 20L of virus inactivation solution for 6 hours.
(4) Removing virus inactivation solution residues: the inactivated placenta is centrifuged to remove the supernatant, and the placenta is washed by adding an equal amount of purified water, and then centrifuged again to remove the supernatant, and the process is repeated for 3 times.
(5) Carrying out three times of circulating freeze thawing treatment on the tissues, wherein the freezing temperature is-60 ℃, and the freezing time is 6 hours; the melting temperature is 25 ℃. After each thaw, the supernatant was discarded by centrifugation and an equal amount of purified water was added again for freezing.
(6) A cell removing process: treating the tissue treated in the step 5) with 0.20% trypsin, 0.10% non-ionic surfactant triton solution and 0.11% ionic surfactant solution sodium dodecyl sulfate solution for 90min respectively, and continuously stirring during the treatment.
(7) Removing the residue of the cell removing solution: washing the tissue treated in the step 6) with purified water for 15min, centrifuging, removing the supernatant, and repeating for 4 times.
(8) First freeze drying: placing the tissue treated in the step 7) into a freeze-drying tray, pre-freezing for 6 hours at-48 ℃, performing gradient freezing and vacuum pumping, and increasing the temperature of each gradient by 10 ℃ to obtain the acellular freeze-dried powder.
(9) Freezing and ball milling: ball milling the dried acellular placenta matrix obtained in the step 8) at low temperature. The ball milled tissue was passed through a 30 mesh screen.
(10) Digesting the acellular freeze-dried powder obtained in the step 9) by using an acid enzyme solution.
(11) Adjusting the pH value of the uniform digestion product obtained in the step 10) to 7.0 by using an alkaline solution, pouring the uniform digestion product into a tray, pre-freezing for 6 hours at the temperature of minus 48 ℃, performing gradient freezing and vacuum pumping, and increasing the temperature of each gradient by 5 ℃ to obtain the acellular porous sponge.
(12) Freezing and ball milling the porous sponge obtained in the step 11) at the temperature of-20 ℃. The ball milled tissue was passed through a 50 mesh screen. To obtain a granular powder with uniform particle size.
(13) And (5) performing irradiation sterilization.
(14) Adding physiological saline into the powder product obtained in the step 13) to prepare a solution with the concentration of 20mg/ml, and shaking up and down for 2min to obtain a uniform gel solution.
(15) And (3) fully mixing the prepared living cells (such as dermal fibroblasts and endometrial mesenchymal stem cells) with the gel liquid (14) and then using the mixture (such as epidermal coverage, subcutaneous injection, uterine cavity injection and the like).
Test of
The extracted human fibroblasts were added to the solution prepared in example one to prepare a gel, which was applied to the subject. And the moisture content of the keratin, the moisture loss rate through the skin, the skin tightness F4 value, the skin elasticity R2 value, the skin smoothness SEsm value and the skin wrinkle SEw value are respectively detected on the day of use, day 7 and day 14.
Wherein, a smaller value of F4 indicates a smoother skin, a larger value of R2 indicates a more elastic skin, a smaller value of SEsm indicates a smoother skin, and a smaller value of SEw indicates a smoother skin. P < 0.05; p < 0.001. As shown in fig. 2, the gel prepared in the first example has improved moisture content, firmness, elasticity, and smoothness of the skin and reduced moisture loss after the subject uses the gel.
The preparation method comprises the steps of extracting endometrial mesenchymal stem cells of a rabbit, adding the extracted endometrial mesenchymal stem cells into a solution prepared in the first embodiment to prepare a gel, injecting the gel into a rabbit uterus with uterine cavity adhesion, injecting 2mL of the product into the rabbit bilateral uterus, and carrying out pathological section staining after the rabbit is cultured for 7 days and 14 days, wherein as shown in figure 3, compared with a uterine cavity adhesion group (Cbutontrol), the gel (Hydrogel) is found to increase the number of glands of endometrium, inhibit uterine fibrosis and improve endometrial receptivity after being injected. Effectively improving the adhesion of the uterine cavity.
Wherein, A picture is HE staining and is used for observing the quantity of endometrial glands, B picture is Masson staining, C picture is TGF-beta 1 immunohistochemical staining, and B picture and C picture are used for observing the uterine fibrosis degree. And D is a scanning electron microscope image. Endometrial receptivity was observed.
Therefore, the preparation and application of the placenta acellular matrix gel loaded with the living cells are adopted, the prepared gel can load various cells and support the growth of the cells, the gelling time is short, and the vitality and the death rate of the cells can be improved.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting the same, and although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: modifications and equivalents may be made to the invention without departing from the spirit and scope of the invention.
Claims (7)
1. The preparation method of the placenta acellular matrix gel loaded with the living cells is characterized by comprising the following steps:
s1, pretreatment of tissues: selecting fresh placenta delivered within 6h, cleaning blood stain with clear water, soaking in sterile storage solution containing peroxyacetic acid and ethanol for 0.5-4h, and storing placenta tissue at-8 deg.C for a long time;
s2, inactivating viruses of the placenta tissues: adding 20L virus inactivating solution into per kilogram of placenta tissue, and treating for 5-10 hr;
s3, removing the virus inactivation solution residues: centrifuging the inactivated placenta, removing supernatant, adding purified water with the same amount for cleaning, centrifuging again, removing supernatant, and repeating for 2-3 times;
s4, carrying out three times of circulating freeze-thaw treatment on the tissue obtained in the step S3;
s5, cell removal treatment: treating the tissue treated in the step S4 with protease solution, nonionic surfactant solution and ionic surfactant solution for 90-120min, wherein stirring is continued;
s6, removing the cell removing solution residue: washing the tissue treated in the step S5 with purified water for 15-30min, centrifuging, removing supernatant, and repeating for 4-6 times;
s7, drying and ball-milling the tissue processed in the step S6, and then performing irradiation sterilization;
s8, adding normal saline into the sterilized powder obtained in the step S7 to dissolve the powder, adding the extracted living cells, uniformly mixing, placing the mixture into different molds, and placing the molds into an incubator at 37 ℃ for 10-20min to form gel.
2. The preparation of a viable cell loaded placental acellular matrix gel according to claim 1, wherein: in step S1, the placenta is transported by cold chain transport at a temperature below 0 ℃.
3. The preparation of a viable cell loaded placental acellular matrix gel according to claim 1, wherein: in step S2, the virus inactivation solution is prepared from 1% -3% peracetic acid, absolute ethanol, and purified water in a volume ratio of 3: 5: 92 to mix.
4. The preparation of a viable cell loaded placental acellular matrix gel according to claim 1, wherein: in step S4, the three-cycle freeze-thaw treatment process includes: the freezing temperature is-80 ℃ to-40 ℃, and the freezing time is not less than 10 hours; the melting temperature is 20-28 ℃, after each melting, the supernatant is centrifuged and discarded, and the same amount of purified water is added again for freezing.
5. The preparation of a viable cell loaded placental acellular matrix gel according to claim 1, wherein: the protease solution in step S5 is 0.20% -0.25% trypsin,
the non-ionic surfactant solution is 0.10-0.15% triton solution,
the ionic surfactant solution is 0.10-0.15% sodium dodecyl sulfate.
6. The preparation of the placenta acellular matrix gel loaded with living cells of claim 1, wherein in step S7, the drying and ball milling process comprises:
first freeze drying: placing the tissue in a freeze-drying tray, pre-freezing for 6-8h at-60- -48 ℃, and carrying out gradient freezing and vacuum pumping, wherein the temperature of each gradient is increased by 10 ℃ to obtain acellular freeze-dried powder;
first freezing ball milling: ball milling the dried acellular placenta matrix at low temperature, and passing the ball-milled tissue through a 30-mesh sieve;
and (3) second freeze drying: digesting the cell-free freeze-dried powder obtained after ball milling with an acid enzyme solution, adjusting the pH value to 6.0-7.0 with an alkaline solution, pouring into a tray, pre-freezing for 6-8 hours at-60 to-48 ℃, performing gradient freezing and vacuum pumping, and raising the temperature of each gradient by 5 ℃ to obtain cell-free porous sponge;
and (3) performing second freezing ball milling: the obtained porous sponge is subjected to freezing ball milling at the temperature of-20 ℃ to-50 ℃, and the ball-milled tissue passes through a 50-mesh sieve to obtain particle powder with uniform particle size.
7. Application of placenta acellular matrix gel loaded with living cells in medical beauty, face-lifting and female health is provided.
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