Culture method for efficiently separating autologous skin source fibroblasts

Abstract

The invention relates to the field of cell culture, in particular to a culture method for efficiently separating autologous skin source fibroblasts, which comprises the following steps: S101, obtaining skin tissue fragments; s102, obtaining a first mixture; s103, obtaining a second mixture; s104, obtaining a third mixture; s105, obtaining a fourth mixture; s106, obtaining a cell precipitate; and S107, performing precipitation and resuspension on the cells by using a fibroblast selective culture medium to obtain a cell suspension, thereby obtaining high-purity fibroblasts. The separation method and the fibroblast selective culture medium provided by the invention can synergistically ensure efficient separation of the fibroblasts and maintenance of high purity and high activity in the culture period, so that the culture period of the fibroblasts is shortened, and the separation method and the fibroblast selective culture medium show a wide application scene in the aspects of beauty, aging resistance, regenerative medicine and the like.

Description

Technical Field

[0001] The invention relates to the field of cell culture, and in particular to a method for efficiently separating and culturing fibroblasts derived from autologous skin. Background Art

[0002] Skin, as the largest organ of the human body, is mainly composed of epidermis, dermis and subcutaneous tissue. Among them, fibroblasts are widely distributed in the dermis. Fibroblasts secrete a variety of substances (such as collagen, elastin, hyaluronic acid, etc.) to form the extracellular matrix of connective tissue and play a structural function for the body. Many studies have shown that fibroblasts isolated from skin tissue have a high proliferation potential and are widely used in beauty, anti-aging and regenerative medicine.

[0003] The existing methods for separating fibroblasts are generally divided into two types: tissue block adhesion method and enzyme digestion method. The tissue block adhesion method is to directly cut the tissue block into pieces and place it on a culture dish for culture. Although the operation is relatively simple, due to the presence of the culture medium, it is difficult for the tissue block to adhere to the culture dish during the adhesion process, resulting in the suspension of the tissue block and the inability of the fibroblasts to crawl out, resulting in a low yield of fibroblasts. Compared with the tissue block adhesion method, the enzyme digestion method obtains a larger number of cells, and the entire cell culture cycle is also shorter than the tissue block adhesion culture method. However, since a single enzyme is used for digestion, the digestion time is long, the activity of fibroblasts is significantly affected, and the cell state is poor. Secondly, the single enzyme is usually pancreatic enzyme, which seriously affects the activity of fibroblasts, making their long-term culture state poor. Finally, a single enzyme can often only decompose a certain type of protein, and there are many types of structural proteins in skin tissue, so a single enzyme often cannot effectively separate fibroblasts. Summary of the invention

[0004] The present invention provides a method for efficiently separating and culturing fibroblasts derived from autologous skin, characterized in that the method comprises the following steps: S101: Cutting the pretreated skin tissue into pieces into predetermined sizes to obtain skin tissue pieces; In some embodiments, the skin tissue is intact skin tissue, which includes three layers: epidermis, dermis and subcutaneous tissue.

[0005] In some embodiments, the skin tissue comprises postauricular skin tissue.

[0006] In some embodiments, the pretreatment includes soaking the collected skin tissue in a first treatment solution and washing it.

[0007] In some embodiments, the first treatment solution comprises IMDM medium and antibiotics.

[0008] In some embodiments, the antibiotic comprises penicillin-streptomycin.

[0009] In some embodiments, the predetermined size includes 1 to 10 mm. 3 .

[0010] S102 placing the skin tissue fragments in a digestive enzyme complex solution to obtain a first mixture; In some embodiments, the digestive enzyme complex solution comprises the following components: 1-10 mg / mL collagenase I, 0.01-0.1 mg / mL collagenase II, 0.01-0.1 mg / mL dispase II, 0.1-1 mg / mL deoxyribonuclease I, 0.1-1 mg / mL hyaluronidase, 0.1-1 mg / mL papain and 0.1-1 mg / mL elastase.

[0011] S103: Ultrasonicate the first mixture using an ultrasonic cleaner to obtain a second mixture; wherein the power of the ultrasonic cleaner is 200-400 W, and the ultrasonication time is 10-15 min.

[0012] In some embodiments, the frequency of the ultrasonic cleaner includes 20~40kHZ.

[0013] In some embodiments, after placing the skin tissue fragments in the digestive enzyme complex solution, step S103 is immediately performed.

[0014] S104 mixing the second mixture to obtain a third mixture; In some embodiments, the mixing comprises oscillating the second mixture for 30 to 60 minutes.

[0015] S105 adding a neutralization medium to the third mixture to terminate the digestion, thereby obtaining a fourth mixture; In some embodiments, the neutralization medium comprises IMDM medium and fetal bovine serum.

[0016] In some embodiments, the ratio of the volumes of the third mixture and the neutralization medium comprises 1:2.

[0017] S106 centrifuging the fourth mixture to obtain a cell pellet; S107 Resuspending the cell pellet in a fibroblast selection medium to obtain a cell suspension, and culturing the cell suspension under predetermined conditions to obtain high-purity fibroblasts.

[0018] In some embodiments, the fibroblast selection medium comprises at least the following components: IMDM medium, stem cell extract, arginine, transforming growth factor β, lipopolysaccharide, retinoic acid and soluble calcium chloride.

[0019] In some embodiments, the fibroblast selection medium further comprises one or more of fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, hyaluronic acid, vitamin B5 and recombinant human insulin.

[0020] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, recombinant human insulin, stem cell extract, arginine, hyaluronic acid, transforming growth factor β, vitamin B5, lipopolysaccharide, retinoic acid and soluble calcium chloride.

[0021] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, 1-10% (volume percentage) stem cell extract, 1-10 mM arginine, 1-5 ng / mL transforming growth factor β, 0.1-2 μg / mL lipopolysaccharide, 0.5-5 nM retinoic acid and 1-5 mM soluble calcium chloride.

[0022] In some embodiments, the fibroblast selection medium further comprises one or more of the following components: 10% (volume percentage) fetal bovine serum, 0.5-5 ng / mL epidermal growth factor, 0.1-1 μg / mL hydrocortisone, 0.05-0.5 mM ascorbic acid, 0.1-1 mg / mL hyaluronic acid, 1-10 μg / mL recombinant human insulin and 1-10 μg / mL vitamin B5.

[0023] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, 10% (volume percentage) fetal bovine serum, 0.5-5 ng / mL epidermal growth factor, 0.1-1 μg / mL hydrocortisone, 0.05-0.5mM ascorbic acid, 1-10 μg / mL recombinant human insulin, 1-10% (volume percentage) stem cell extract, 1-10 mM arginine, 0.1-1 mg / mL hyaluronic acid, 1-5ng / mL transforming growth factor β, 0.1-2 μg / mL lipopolysaccharide, 1-10 μg / mL vitamin B5, 0.5-5 nM retinoic acid and 1-5 mM soluble calcium chloride.

[0024] In some embodiments, the method further comprises S108 digesting and passage the fibroblasts when the cell confluence reaches 80-90%.

[0025] In some embodiments, the fibroblasts are greater than 99% pure.

[0026] In some embodiments, the fibroblasts are cultured to passage P4.

[0027] Compared with the prior art, the beneficial technical effects of the present invention include at least the following aspects: Complete skin tissue includes three layers: epidermis, dermis and subcutaneous tissue, and skin fibroblasts are the main cells of dermis. Therefore, in order to obtain high-purity skin fibroblasts, the prior art usually separates fibroblasts by taking dermis alone (for example, directly separating dermis tissue (for example, Chinese patent application CN110438067A), and using dispase to separate epidermis and dermis in advance (for example, Chinese patent application CN118406636A)).

[0028] The present invention breaks through the conventional thinking of the prior art and creatively proposes a one-step digestion separation method of intact skin tissue using a digestive enzyme complex solution + ultrasonic cavitation (i.e., ultrasonic enzymolysis). It should be emphasized that compared with visceral tissues (such as heart, liver, kidney, fat, etc.), the structure of skin tissue is more compact and conventional separation is more difficult. In addition, the prior art also believes that the use of high-concentration enzymes (or enzymes with complex components) or high-intensity ultrasound alone will damage the activity of cells (such as fibroblasts). The present invention found that the separation method provided by the present invention (i.e., the combination of digestive enzyme complex solution + ultrasonic cavitation) not only has no adverse effect on the activity of fibroblasts, but also unexpectedly, compared with the existing method for separating skin fibroblasts (usually more than 30 minutes), the present invention significantly shortens the speed of skin dissociation, and only takes 10-15 minutes to obtain a high number of fibroblasts with high activity, which greatly increases the separation efficiency and cell yield of fibroblasts, while also synergistically promoting the proliferation rate and cell number of fibroblasts in culture, shortening the culture cycle of fibroblasts.

[0029] It should be further explained that although the separation method provided by the present invention can increase the number of isolated fibroblasts to a certain extent, as more cells are separated, epidermal cells are inevitably separated, which may cause epidermal cell contamination and interfere with the subsequent culture purity and expansion of fibroblasts. The present invention found that after the cells separated by the present invention were cultured with a fibroblast selection medium, at the P1 generation (or after the primary cells were fully spread), epidermal cells were almost absent therein, and the fibroblasts obtained by the present invention can be maintained in a good state for a long time. In other words, the fibroblast selection medium provided by the present invention can inhibit the growth of miscellaneous cells while not affecting or even promoting the growth of fibroblasts.

[0030] Furthermore, the separation method and fibroblast selection culture medium provided by the present invention can synergistically ensure the efficient separation of fibroblasts and the maintenance of high purity and high activity during culture, thereby shortening the primary fibroblast culture cycle, and can stably carry out multiple primary fibroblast cultures. It is easy to operate and highly practical, and has a wide range of application scenarios in beauty, anti-aging, and regenerative medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0032] Figure 1 This is a picture showing the morphological results of cells obtained by ultrasonic enzymatic separation in Preparation Example 1; Figure 2 This is a morphological result diagram of the primary cells isolated in Preparation Example 1 after 1-2 days of culture; Figure 3 This is a morphological result diagram of the primary cells isolated in Preparation Example 1 when cultured for 5 to 7 days; Figure 4 This is a morphological result diagram of the P3 generation cells of Preparation Example 1; Figure 5 This is the CD104-result diagram of the P4 generation cells of Preparation Example 1; Figure 6 This is a graph showing the CD90+ results of P4 cells in Preparation Example 1. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0036] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0037] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0038] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0039] Embodiment 1 This embodiment provides a method for efficiently isolating and culturing fibroblasts from autologous skin, and the specific steps are as follows: S101: Cutting the pretreated skin tissue into pieces into predetermined sizes to obtain skin tissue pieces; In some embodiments, the skin tissue is intact skin tissue, which includes three layers: epidermis, dermis and subcutaneous tissue.

[0040] In some embodiments, the skin tissue comprises postauricular skin tissue.

[0041] In some embodiments, the pretreatment includes soaking the collected skin tissue in a first treatment solution and washing it.

[0042] In some embodiments, the first treatment solution comprises IMDM medium and antibiotics.

[0043] In some embodiments, the antibiotic comprises penicillin-streptomycin.

[0044] In some embodiments, the predetermined size includes 1 to 10 mm. 3 .

[0045] S102 placing the skin tissue fragments in a digestive enzyme complex solution to obtain a first mixture; In some embodiments, the digestive enzyme complex solution comprises the following components: 1-10 mg / mL collagenase I, 0.01-0.1 mg / mL collagenase II, 0.01-0.1 mg / mL dispase II, 0.1-1 mg / mL deoxyribonuclease I, 0.1-1 mg / mL hyaluronidase, 0.1-1 mg / mL papain and 0.1-1 mg / mL elastase.

[0046] S103 ultrasonically clean the first mixture using an ultrasonic cleaning apparatus to obtain a second mixture; wherein the power of the ultrasonic cleaning apparatus is 200-400 W, and the ultrasonic time is 10-15 min; In some embodiments, the frequency of the ultrasonic cleaner includes 20~40kHZ.

[0047] In some embodiments, after placing the skin tissue fragments in the digestive enzyme complex solution, step S103 is immediately performed.

[0048] S104 mixing the second mixture to obtain a third mixture; In some embodiments, the mixing comprises oscillating the second mixture for 30 to 60 minutes.

[0049] S105 adding a neutralization medium to the third mixture to terminate the digestion, thereby obtaining a fourth mixture; In some embodiments, the ratio of the volumes of the third mixture and the neutralization medium comprises 1:2.

[0050] In some embodiments, the neutralization medium comprises IMDM medium and fetal bovine serum.

[0051] S106 centrifuging the fourth mixture to obtain a cell pellet; S107 Resuspending the cell pellet in a fibroblast selection medium to obtain a cell suspension, and culturing the cell suspension under predetermined conditions to obtain high-purity fibroblasts.

[0052] In some embodiments, the predetermined conditions include culturing in an incubator at 37° C. and 5% carbon dioxide.

[0053] In some embodiments, the fibroblast selection medium comprises at least the following components: IMDM medium, stem cell extract, arginine, transforming growth factor β, lipopolysaccharide, retinoic acid and soluble calcium chloride.

[0054] In some embodiments, the fibroblast selection medium further comprises one or more of fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, hyaluronic acid, vitamin B5 and recombinant human insulin.

[0055] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, recombinant human insulin, stem cell extract, arginine, hyaluronic acid, transforming growth factor β, vitamin B5, lipopolysaccharide, retinoic acid and soluble calcium chloride.

[0056] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, 1-10% (volume percentage) stem cell extract, 1-10 mM arginine, 1-5 ng / mL transforming growth factor β, 0.1-2 μg / mL lipopolysaccharide, 0.5-5 nM retinoic acid and 1-5 mM soluble calcium chloride.

[0057] In some embodiments, the fibroblast selection medium further comprises one or more of the following components: 10% (volume percentage) fetal bovine serum, 0.5-5 ng / mL epidermal growth factor, 0.1-1 μg / mL hydrocortisone, 0.05-0.5 mM ascorbic acid, 0.1-1 mg / mL hyaluronic acid, 1-10 μg / mL recombinant human insulin and 1-10 μg / mL vitamin B5.

[0058] In some embodiments, the fibroblast selection medium comprises the following components: IMDM medium, 10% (volume percentage) fetal bovine serum, 0.5-5 ng / mL epidermal growth factor, 0.1-1 μg / mL hydrocortisone, 0.05-0.5mM ascorbic acid, 1-10 μg / mL recombinant human insulin, 1-10% (volume percentage) stem cell extract, 1-10 mM arginine, 0.1-1 mg / mL hyaluronic acid, 1-5ng / mL transforming growth factor β, 0.1-2 μg / mL lipopolysaccharide, 1-10 μg / mL vitamin B5, 0.5-5 nM retinoic acid and 1-5 mM soluble calcium chloride.

[0059] In some embodiments, the method further comprises S108 digesting and passage the fibroblasts when the cell confluence reaches 80-90%.

[0060] In some embodiments, the fibroblasts are greater than 99% pure.

[0061] Preparation Example 1 In the operating room, 1 to 5 pieces of skin biopsy behind the ear (provided by Hainan Boao Future Hospital) were taken and stored in a sterile antibiotic-containing culture medium (IMDM culture medium, 1% PS (penicillin-streptomycin, volume percentage), the same below), and transported to the laboratory using a cold chain. On a million-level clean bench in a GMP-grade laboratory, the skin tissue was washed three times with an antibiotic-containing culture medium (IMDM culture medium, 1% PS). Then, the skin tissue was chopped into 1 mm pieces with scissors. 3 The fragments were washed 3-5 times with IMDM washing solution (IMDM culture medium, 1% PS), transferred to a centrifuge tube containing 20 mL of digestive enzyme complex solution, placed in an ultrasonic cleaner for ultrasonication, and then placed in a 37°C constant temperature shaker for 30-60 minutes.

[0062] The components of the digestive enzyme complex solution specifically include: collagenase I 5 mg / mL; collagenase II 0.1 mg / mL; dispase II 0.1 mg / mL; deoxyribonuclease I 1 mg / mL; hyaluronidase 0.1 mg / mL; papain 0.1 mg / mL; and elastase 0.1 mg / mL.

[0063] The ultrasonic method specifically includes: placing in an ultrasonic cleaner for 10 minutes; during this period, the water bath in the ultrasonic cleaner is kept at a constant temperature of 37° C., the power of the ultrasonic cleaner is set to 200W, and the frequency is set to 20-40kHZ.

[0064] After the shaking and mixing, add 2 times the volume of neutralization medium (IMDM medium, 10% FBS (fetal bovine serum, volume percentage)) and mix well, then centrifuge for 10 minutes, discard the supernatant, and add 10 mL of fibroblast selection medium to mix. After resuspending, add to T25 cell culture flask and culture in a 37°C, 5% carbon dioxide incubator until 80-90%, with multiple half-medium changes and subcultures during this period.

[0065] The components of the fibroblast selection medium specifically include: IMDM medium; 10% (volume percentage) of fetal bovine serum; 0.5 ng / mL of epidermal growth factor; 0.1 μg / mL of hydrocortisone; 0.05 mM of ascorbic acid; 1 μg / mL of recombinant human insulin; 1% (volume percentage) of stem cell extract; 10 mM of arginine; 0.1 mg / mL of hyaluronic acid; 5 ng / mL of transforming growth factor β; 10 μg / mL of vitamin B5; 2 μg / mL of lipopolysaccharide; 5 nM of retinoic acid; and 5 mM of soluble calcium chloride.

[0066] The stem cell extract is the supernatant of a healthy mesenchymal stem cell (eg, P3) culture.

[0067] Figure 1 The results showed that the cells obtained by ultrasonic enzymatic separation in this preparation example were distributed as single cells, without large cell clusters or fine tissue blocks.

[0068] Figure 2 The results showed that after 1 to 2 days of culture, primary cells (P0 generation) began to grow adherently and had a relatively simple morphology.

[0069] Figure 3 The results showed that after 5 to 7 days of culture, the primary cells had covered the entire cell culture flask, and the cell morphology was single, showing a typical fibroblast-like spindle shape.

[0070] Figure 4 The results showed that the cells after the second passage (P3 generation) grew and multiplied rapidly, and the cells reached a density of 90% in about 3 to 5 days of culture. Moreover, the cells still maintained a single morphology, showing a typical spindle shape, with an oval nucleus in the center, cytoplasmic protrusions, and radial growth without the growth of any miscellaneous cells.

[0071] Fibroblast surface marker detection experiment 1) Take the culture flask of P4 fibroblasts prepared in Preparation Example 1 and wash it twice with physiological saline.

[0072] 2) Add 0.25% trypsin solution to the culture flask cleaned in step 1), digest at 37°C for 3 minutes, and then terminate the digestion with ordinary culture medium to obtain a cell suspension.

[0073] 3) Place the cell suspension obtained in step 2) into a 15 mL centrifuge tube, add physiological saline, and centrifuge at 1200 rpm for 5 min; discard the supernatant, and repeat the above steps 3 times.

[0074] 4) Take four 1.5 mL EP centrifuge tubes and label all the antibody names and fluorescein; use a micropipette to draw the sample from step 3) and add 5×10 5 Then add PBS to the above centrifuge tubes to make the total volume of each tube 1 mL.

[0075] 5) Place the centrifuge tube in a vortex mixer and mix for 3 seconds. Centrifuge each sample tube at 3000rpm for 5 minutes, discard the supernatant and recover the cells. Add 1mL PBS and repeat the above steps once. Then add 100μL PBS to the above four tubes and resuspend the recovered cells.

[0076] 6) According to the label of the sample tube, add appropriate amount of corresponding fluorescent antibodies to the four sample tubes: IgG1-FITC, IgG2a-PE, CD90-FITC (fibroblast marker), CD104-PE (epidermal cell marker). After mixing, incubate at 2-8℃ in the dark for 30 minutes.

[0077] 7) Wash the cells with an appropriate amount of PBS, centrifuge at 3000rpm for 5 minutes, and discard the supernatant. Finally, add 350μL PBS to each tube to resuspend the cells.

[0078] 8) Analyze the resuspended cells using flow cytometry.

[0079] The experimental results of Preparation Example 1 show that the present invention can increase the number of fourth-generation fibroblasts to more than 1×10 within 30 days without the need for a special bioreactor. 8 cells (see Table 1 for details), which fully meets the clinical application. Figure 5 and Figure 6 The results showed that the CD90+ and CD104- of P4 generation fibroblasts were greater than 99% when surface markers were detected by flow cytometry, which indicated that the fibroblasts obtained in this preparation example had high purity and strong primary activity.

[0080] Preparation Example 2 The difference between this preparation example and preparation example 1 is that the concentration of collagenase I in the digestive enzyme complex solution is 1 mg / mL, the concentration of dispase is 0.01 mg / mL, the concentration of hyaluronidase is 1 mg / mL, and the concentrations of the other enzymes are the same as those in preparation example 1. In addition, the power of the ultrasonic cleaner is set to 400 W. Finally, the concentration of the stem cell extract in the fibroblast selection medium used is 10% (volume percentage), the concentration of arginine is 1 mM, the concentration of hyaluronic acid is 1 mg / mL, the concentration of retinoic acid is 0.5 mM, the concentration of soluble calcium chloride is 1 mM, and the concentrations of the other components are the same as those in preparation example 1.

[0081] The subsequent culture steps were the same as those in Preparation Example 1. The P4 cell count, purity and culture period are detailed in Table 1.

[0082] Comparative Example 1 The difference between this comparative example and Preparation Example 1 is that when treating skin fragments, only the digestive enzyme complex solution is added without simultaneous ultrasonic treatment. The subsequent culture steps are the same as Preparation Example 1. The P4 cell count, purity and culture period are detailed in Table 1.

[0083] Comparative Example 2 The difference between this comparative example and Preparation Example 1 is that the digestive enzyme complex solution only uses collagenase I, collagenase II and dispase, and the concentration of each enzyme is the same as that of Preparation Example 1. The ultrasonic treatment and subsequent culture steps are the same as those of Preparation Example 1. The P4 cell count, purity and culture period are detailed in Table 1.

[0084] Comparative Example 3 The difference between this comparative example and Preparation Example 1 is that the concentration of collagenase II in the digestive enzyme complex solution is adjusted to 0.5 mg / mL, the concentration of dispase is adjusted to 0.5 mg / mL, and the concentration of deoxyribonuclease I is adjusted to 0.05 mg / mL. The ultrasonic treatment and subsequent culture steps are the same as those in Preparation Example 1. The P4 cell count, purity and culture period are detailed in Table 1.

[0085] Comparative Example 4 The difference between this comparative example and Preparation Example 1 is that the concentration of arginine in the fibroblast selection medium is adjusted to 0.1 mM; the concentration of stem cell extract is adjusted to 0.1%; and the concentration of lipopolysaccharide is adjusted to 5 μg / mL. Ultrasonic treatment and subsequent culture steps are the same as those in Preparation Example 1. The P4 cell count, purity, and culture period are detailed in Table 1.

[0086] Comparative Example 5 The difference between this comparative example and Preparation Example 1 is that the concentration of transforming growth factor β in the fibroblast selection medium is adjusted to 10 ng / mL; the concentration of soluble calcium chloride is adjusted to 10 mM. The ultrasonic treatment and subsequent culture steps are the same as those in Preparation Example 1. The P4 cell count, purity and culture period are detailed in Table 1.

[0087] Comparative Example 6 The difference between this comparative example and Preparation Example 1 is that in the culture process of the isolated fibroblasts, IMDM medium is used instead of fibroblast selection medium, and the subsequent culture steps are the same as those in Preparation Example 1. The P4 cell count, purity and culture period are shown in Table 1.

[0088] Table 1 The results are shown in Table 1. Compared with the results of Comparative Examples 1-6, the results of Preparation Examples 1 and 2 show that the method provided by the present invention can not only efficiently separate fibroblasts, but also the separated fibroblasts have a fast proliferation rate, high purity, and more cells, which is more conducive to subsequent applications in cosmetic medical treatment and other projects and reduces the waiting time of patients. The results of Comparative Example 1 show that if only the digestive enzyme complex solution provided by the present invention is used without ultrasonic treatment, the proliferation rate and cell number of fibroblasts will be seriously affected, so that the time to reach the P4 generation will be significantly extended. The results of Comparative Example 6 show that if the fibroblast selection medium provided by the present invention is not used, the proliferation rate, cell number and purity of fibroblasts will be affected at the same time, and the time to reach the P4 generation will be significantly extended. The results of Comparative Examples 2 and 3 show that if some components of the digestive enzyme complex solution (for example, only some digestive enzymes) and concentrations are adjusted, the proliferation rate and cell number (even purity) of fibroblasts will also be affected to a certain extent, so that the time to reach the P4 generation will be significantly extended. The results of Comparative Examples 4 and 5 show that if the concentration of some components of the fibroblast selection medium is adjusted, the proliferation rate and cell number (even purity) of fibroblasts will be affected to a certain extent, so that the time to reach the P4 generation is significantly prolonged.

[0089] Embodiment 2 This embodiment provides an autologous cosmetic biomaterial prepared by the method provided in the first embodiment, wherein the autologous cosmetic biomaterial comprises autologous isolated and cultured fibroblasts, physiological saline, a mixed solution rich in compound amino acids, stem cell-related growth factors, etc. The number of the fibroblasts is about 1.0×10 8 / mL, purity greater than 99%, activity greater than 98%.

[0090] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the enlightenment of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the purpose of the present invention and the claims, which all fall within the protection of the present invention.

Claims

1. A method for efficiently isolating and culturing fibroblasts derived from autologous skin, characterized in that: The following steps are involved: S101: Cutting the pretreated skin tissue into pieces into predetermined sizes to obtain skin tissue pieces; S102 placing the skin tissue fragments in a digestive enzyme complex solution to obtain a first mixture; S103 ultrasonically clean the first mixture using an ultrasonic cleaning apparatus to obtain a second mixture; wherein the power of the ultrasonic cleaning apparatus is 200-400 W, and the ultrasonic time is 10-15 min; S104 mixing the second mixture to obtain a third mixture; S105 adding a neutralization medium to the third mixture to terminate the digestion, thereby obtaining a fourth mixture; S106 centrifuging the fourth mixture to obtain a cell pellet; S107 resuspending the cell pellet using a fibroblast selection medium to obtain a cell suspension, and culturing the cell suspension under predetermined conditions to obtain high-purity fibroblasts; The digestive enzyme complex solution comprises the following components: 1-10 mg / mL collagenase I, 0.01-0.1 mg / mL collagenase II, 0.01-0.1 mg / mL dispase II, 0.1-1 mg / mL deoxyribonuclease I, 0.1-1 mg / mL hyaluronidase, 0.1-1 mg / mL papain and 0.1-1 mg / mL elastase; the fibroblast selection culture medium comprises at least the following components: IMDM culture medium, stem cell extract, arginine, transforming growth factor β, lipopolysaccharide, retinoic acid and soluble calcium chloride.

2. The method according to claim 1, characterized in that The skin tissue is complete skin tissue, which includes three layers: epidermis, dermis and subcutaneous tissue.

3. The method according to claim 1, characterized in that The fibroblast selection medium also includes one or more of fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, hyaluronic acid, vitamin B5 and recombinant human insulin.

4. The method according to claim 1, wherein: The method further comprises S108 digesting and passage the fibroblasts when the cell confluence reaches 80-90%.

5. The method according to claim 1, wherein The mixing includes oscillating and mixing the second mixture for 30 to 60 minutes.

6. The method according to claim 1, characterized in that The fibroblast selection medium comprises the following components: IMDM medium, fetal bovine serum, epidermal growth factor, hydrocortisone, ascorbic acid, recombinant human insulin, stem cell extract, arginine, hyaluronic acid, transforming growth factor β, vitamin B5, lipopolysaccharide, retinoic acid and soluble calcium chloride.

7. The method according to claim 1, characterized in that The predetermined size includes 1-10 mm 3 .

8. The method according to claim 1, characterized in that The skin tissue includes post-auricular skin tissue.

9. The method according to claim 1, wherein: The fibroblast selection medium comprises the following components: IMDM medium, 1-10% stem cell extract, 1-10 mM arginine, 1-5 ng / mL transforming growth factor β, 0.1-2 μg / mL lipopolysaccharide, 0.5-5 nM retinoic acid and 1-5 mM soluble calcium chloride.

10. The method according to claim 1, characterized in that The purity of the fibroblasts is greater than 99%.

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