Method for rapidly digesting mouse skin tissue into single cells

Abstract

The invention relates to the field of tissue dissociation and cell separation, and discloses a method for rapidly digesting mouse skin tissue into single cells. According to the method, the skin tissue on the back of an adult mouse is taken as an example, different enzyme digestion modes and mechanical treatment modes are combined and used, and the optimal dissociation condition is screened out, so that the skin tissue can be dissociated into the single-cell suspension within a short time. According to the technical scheme, the efficient dissociation of the skin tissue is realized through the optimal combination of the enzyme type, the concentration and the mechanical treatment mode, and the required treatment time is greatly shortened; the cell purity and the cell viability are effectively improved, and an ideal cell sample is provided for subsequent single cell omics research; and reliable technical support is provided for subsequent flow cytometry analysis, cell sorting, high-throughput sequencing and other researches.

Description

Technical Field

[0001] The invention belongs to the field of tissue dissociation and cell separation, and specifically discloses a method for rapidly dissociating skin tissue into a single cell suspension. Background Art

[0002] Skin tissue, as the largest organ in the human body, has a unique structure and function that relies on the rich collagen, elastic fibers and other connective tissue components. These components are intertwined to form a dense and tough network structure, providing the skin with a strong protective barrier and elasticity. However, this dense tissue structure also poses considerable challenges to scientific research, especially research at the single-cell level. Without mechanical assistance, conventional enzymatic digestion processes often take several hours or even longer to effectively separate cells from skin tissue, which not only greatly limits research efficiency, but may also cause changes in the state of cells during long-term processing, affecting the accuracy of subsequent analysis.

[0003] In recent years, with the rapid progress of high-throughput sequencing technology, single-cell sequencing has become an important means to study cellular heterogeneity, developmental trajectories, disease mechanisms and other fields. Especially in skin tissue, single-cell sequencing can help scientists deeply understand the role of different cell types in skin homeostasis maintenance, damage repair, immune response and disease development. Therefore, efficient, rapid and gentle dissociation of skin tissue to obtain high-quality single-cell suspension has become an urgent need in current single-cell sequencing research.

[0004] However, existing skin tissue dissociation technologies face significant challenges. Traditional physical methods such as mechanical grinding and shearing, although they can accelerate the dissociation process, often cause physical damage to cells, affecting cell integrity and vitality. Although the enzyme digestion method is relatively gentle on cells, it is inefficient due to the density of skin tissue and is difficult to meet the dual requirements of timeliness and sample quality for large-scale single-cell sequencing studies. In addition, long-term enzyme treatment may also introduce additional enzyme activity interference, further affecting the accuracy of subsequent sequencing data.

[0005] Therefore, developing new skin tissue dissociation technologies to efficiently and gently dissociate skin tissue into single-cell suspensions in a short period of time has become the key to promoting the in-depth development of skin science research. This requires researchers to continuously explore and optimize dissociation conditions, such as finding more efficient enzyme combinations, developing new biophysical dissociation technologies, or combining advanced technologies such as microfluidics and lasers, in order to significantly improve dissociation efficiency and purity while protecting cell integrity and activity, thereby meeting the urgent needs of downstream omics research, especially single-cell sequencing research, for high-quality samples. Summary of the invention

[0006] In response to the above problems, the present invention discloses a method for quickly digesting mouse skin tissue into single cells. This method can be used for single-cell omics research such as flow cytometry analysis, cell sorting, and high-throughput sequencing, and is particularly suitable for experimental scenarios that require obtaining high-quality single-cell suspensions in a relatively short time.

[0007] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: A method for rapidly digesting mouse skin tissue into single cells comprises the following steps: 1) Pretreatment: Mouse skin tissue was used as the object, hair was removed, and then cut into small pieces under sterile conditions; 2) Digestion: Add enzymatic solution containing type I collagenase to the above small pieces, and perform mechanical constant temperature beating treatment at the same time. The digestion time is ≤15 minutes.

[0008] Furthermore, in the above-mentioned method for rapidly digesting mouse skin tissue into single cells, the mouse is an adult mouse, the skin is back skin, and skin tissue with a diameter of 0.5-1.5 cm, preferably a diameter of 1.0 cm, is taken.

[0009] Furthermore, in the above method for rapidly digesting mouse skin tissue into single cells, the enzymatic solution is prepared by adding type I collagenase and DNase I to Hanks buffer; The concentration of type I collagenase in the enzymatic solution is 1-3 mg / ml, preferably 2 mg / ml; the activity of DNase I is 5-15 U / ml, preferably 10 U / ml.

[0010] Furthermore, in the above-mentioned method for rapidly digesting mouse skin tissue into single cells, the ratio of the amount of the enzymatic hydrolysate to the diameter of the skin tissue is 1-10:1 ml / cm, preferably 5:1 ml / cm.

[0011] Furthermore, in the above-mentioned method for rapidly digesting mouse skin tissue into single cells, the mechanical constant temperature beating treatment includes: constant temperature beating treatment, using a sterile transparent sealing bag, 35-40°C (preferably 37°C) constant temperature reciprocating beating, a frequency of 180-360 times / min (preferably a frequency of 180 times / min); beating using a beating homogenizer.

[0012] Furthermore, the above method for rapidly digesting mouse skin tissue into single cells further comprises: Cell purification: After digestion, hyaluronidase is added to prevent cell agglomeration during centrifugation, and red blood cell lysis buffer is used to remove potential residual red blood cells. Subsequently, a cell filter is used to remove large impurities to obtain tissue sample digestion solution; the tissue sample digestion solution is centrifuged to remove the supernatant containing particle debris, and then resuspended and centrifuged in DMEM culture medium to remove tissue debris. Finally, the cells are resuspended in an appropriate amount of complete culture medium containing serum and observed under a microscope, while the activity of the digestive enzyme is terminated.

[0013] Furthermore, in the above-mentioned method for rapidly digesting mouse skin tissue into single cells, the amount of hyaluronidase added is 0.1-1 mg / ml, preferably 0.5 mg / ml; the cell filter is preferably 30 microns; the centrifugation parameters are 1000-3000 rpm, 2-4 min, preferably 2000 rpm, 3 min; the DMEM culture medium is resuspended and centrifuged 1-5 times, preferably 3 times.

[0014] Furthermore, the above method for rapidly digesting mouse skin tissue into single cells, after the cell purification, further comprises: Cell viability detection: The live cells of the single cell suspension obtained by digestion were counted, and the cell viability was calculated by trypan blue staining.

[0015] The invention also discloses the application of the method in flow cytometric analysis, cell sorting or high-throughput sequencing.

[0016] The present invention has the following beneficial effects: The present invention provides a method for rapidly dissociating skin tissue into a single cell suspension, taking the back skin tissue of adult mice as an example. By combining different enzyme digestion methods and mechanical treatment methods, the optimal dissociation conditions are screened out, so that the skin tissue can be dissociated into a single cell suspension in a relatively short time. The technical solution of the present invention achieves efficient dissociation of skin tissue by optimizing the combination of enzyme types, concentrations and mechanical treatment methods, greatly shortening the required processing time, specifically: 1) Compared with the existing technology, the digestion time is significantly shortened: compared with the enzyme dissociation time of the existing technology (such as 90-180 minutes for pig skin tissue in CN202310137290, 50-90 minutes for mouse skin tissue in CN202410617733, about 60 minutes for human corneal epithelium in CN202410537537, and 70 minutes for skin tissue of unknown origin in CN202110322990), the present invention only takes 15 minutes to complete, greatly improving the experimental efficiency.

[0017] 2) Preservation of cell physiological state: Shortening the in vitro treatment time helps maintain the original state of cells, reduces the impact on cell gene expression and physiological activities, and ensures the biological consistency of single-cell suspensions, which is of great significance for single-cell omics research.

[0018] 3) Efficient digestion and purification process: The use of hyaluronidase and red blood cell lysis buffer, combined with filtration and centrifugation steps, effectively removes residual impurities, ensures high-purity and high-viability single-cell suspension, and provides a more ideal cell sample for subsequent experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Comparison of enzyme digestion effects between groups A and B under constant temperature shaking treatment; Figure 2 Comparison of digestion effects between combination 3 and combination 4 under mechanical constant temperature beating treatment; Figure 3 Trypan blue staining after digestion of combination 3 and combination 4 and microscopic observation of cells of combination 4; Figure 4 The results of flow cytometry sorting of mouse dermal sheath cells labeled with tdTomato are shown; Figure 5 Fluorescence microscopy results of flow-sorted tdTomato-labeled mouse dermal sheath cells; Figure 6 Fluorescence microscopy examination of the adherent growth of sorted tdTomato-labeled dermal sheath cells in vitro. DETAILED DESCRIPTION

[0020] A method for rapidly digesting mouse skin tissue into single cells comprises the following steps: 1) Pretreatment: Mouse skin tissue was used as the object, hair was removed, and then cut into small pieces; 2) Digestion: Add enzymatic solution containing type I collagenase to the above small pieces, and perform mechanical constant temperature beating treatment at the same time. The digestion time is ≤15 minutes.

[0021] The mouse is an adult mouse, the skin is back skin, and skin tissue with a diameter of 0.5-1.5 cm, preferably 1.0 cm in diameter, is obtained.

[0022] The enzymatic solution is prepared by adding type I collagenase and DNase I into Hanks buffer; The concentration of type I collagenase in the enzymatic solution is 1-3 mg / ml, preferably 2 mg / ml; the activity of DNase I is 5-15 U / ml, preferably 10 U / ml.

[0023] The ratio of the amount of the enzymatic solution to the diameter of the skin tissue is 1-10:1 ml / cm, preferably 5:1 ml / cm.

[0024] The mechanical constant temperature beating treatment includes: constant temperature beating treatment, using a sealing bag, constant temperature reciprocating beating at 35-40°C (preferably 37°C), a frequency of 180-360 times / min (preferably 180 times / min); beating using a beating homogenizer.

[0025] After digestion is complete, it also includes: 3) Cell purification: After digestion, hyaluronidase is added to prevent cell clumping during centrifugation, and red blood cell lysis buffer is used to remove potential residual red blood cells. Subsequently, a cell filter is used to remove large impurities to obtain tissue sample digestion solution; the tissue sample digestion solution is centrifuged to remove the supernatant containing particle debris, and then resuspended and centrifuged in DMEM culture medium to remove tissue debris. Finally, the cells are resuspended in an appropriate amount of complete culture medium containing serum and observed under a microscope, while the activity of the digestive enzyme is terminated.

[0026] The amount of hyaluronidase added is 0.1-1 mg / ml, preferably 0.5 mg / ml; the cell filter is preferably 30 microns; the centrifugation parameters are 1000-3000 rpm, 2-4 min, preferably 2000 rpm, 3 min; the DMEM culture medium is resuspended 1-5 times, preferably 3 times.

[0027] After cell purification, it also includes: 4) Cell viability detection: Live cells were counted in the single-cell suspension obtained by digestion, and the cell viability was calculated by trypan blue staining.

[0028] After orthogonal test verification, the following specific parameters are selected: Type I collagenase (Solarbio, C8140) 2 mg / ml, DNase I (Solarbio, D8071) 10 U / ml, prepared using Hanks buffer (containing calcium and magnesium ions, Solarbio, H1020), 5 ml was used for one set of samples.

[0029] Mechanical treatment method 2: Constant temperature beating treatment, using a sterile transparent sealing bag, 37°C constant temperature reciprocating beating, frequency 180 times / min. Instrument: beating homogenizer, Shanghai Zigui Instrument Co., Ltd., model ZGJZQ-10JM.

[0030] 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. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0031] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example

[0033] 1. Material preparation: The dorsal skin tissue of adult C57BL / 6 mice was used as the object. After the hair was cut off, the skin tissue with a diameter of 1 cm was obtained and cut into small pieces for later use.

[0034] 2. Digestion and mechanical treatment grouping: (1) Enzyme digestion method: Group A: 2 mg / ml collagenase type I (Solarbio, C8140), 10 U / ml DNase I (Solarbio, D8071), prepared using Hanks buffer (containing calcium and magnesium ions, Solarbio, H1020), 5 ml was used for one group of samples.

[0035] Group B: 2 mg / ml collagenase type II (Solarbio, C8150), 0.25% trypsin (Solarbio, T1360), 10 U / ml DNase I (Solarbio, D8071), also prepared using Hanks buffer (containing calcium and magnesium ions, Solarbio, H1020), 5 ml was used for one group of samples.

[0036] (2) Mechanical treatment method: Mechanical treatment method 1: Constant temperature shaking treatment, using 15 ml centrifuge tubes, 37°C constant temperature gyratory shaking, frequency 220 times / min. Instrument: Constant temperature water bath shaker (Tianjin Sadelis Laboratory Analysis Instrument Manufacturing Factory, model SHA-B).

[0037] Mechanical treatment method 2: Constant temperature beating treatment, using a sterile transparent sealing bag, 37°C constant temperature reciprocating beating, frequency 180 times / min. Instrument: beating homogenizer, Shanghai Zigui Instrument Co., Ltd., model ZGJZQ-10JM.

[0038] (3) Experimental combination: The two enzymatic digestion methods and the two mechanical treatment methods were combined in pairs, resulting in four combinations: Combination 1: Enzyme digestion method A + mechanical treatment method 1; Combination 2: Enzyme digestion method B + mechanical treatment method I; Combination 3: Enzyme digestion method B + mechanical treatment method II; Combination 4: Enzyme digestion method A + mechanical treatment method II.

[0039] Test Example 1

[0040] The technical solution of Example 1 was tested.

[0041] 1) Comparison of digestion results: Combination 1 and combination 2 (constant temperature shaking treatment) showed almost no obvious digestion effect after 4 hours; only a small amount of tissue dissociation was observed after 20 hours, and the digestion was incomplete, indicating that constant temperature shaking treatment could not quickly digest skin tissue ( Figure 1 ).

[0042] Combination 3 and combination 4 (mechanical constant temperature patting treatment) were compared after 15 minutes. The experimental results show that ( Figure 2 ), the skin tissue of combination 3 still has a lot of flocculent residues and is not completely digested, while the skin tissue of combination 4 has been basically completely digested and is in a single-cell state, with a significant dissociation effect.

[0043] 2) Trypan blue staining and microscopic examination: The tissue samples after digestion of combination 3 and combination 4 were observed under a microscope using trypan blue staining. The results showed that a large amount of undigested mass tissue and collagen fiber fragments remained in combination 3 ( Figure 3 -a, b, while only a small amount of collagen fiber fragments remained in combination 4 ( Figure 3 -c), indicating that the digestion effect of combination 4 is better.

[0044] 3) Cell purification and cell viability detection: For combination 4, after digestion, 0.5 mg / ml hyaluronidase (Solarbio, H8030) was added to prevent cell clumping during centrifugation, and red blood cell lysis buffer (Solarbio, R1010) was used to remove potential residual red blood cells, followed by a 30-micron cell filter (pluriStrainer, SKU 43-10030-40) to remove large impurities. The tissue sample digestion solution was centrifuged at 2000 rpm for 3 minutes to remove the supernatant, and resuspended in DMEM medium and repeated three times to remove more than 99% of tissue debris. Finally, the cells were resuspended in an appropriate amount of complete medium containing serum and observed under a microscope ( Figure 3-d), while terminating the activity of digestive enzymes.

[0045] The live cells in the single cell suspension obtained by digestion were counted. The cell survival rate was calculated by trypan blue staining, and the average survival rate was 92.3%. This method effectively improved the cell purity and cell viability, providing an ideal cell sample for subsequent single cell omics research.

[0046] Test Example 2

[0047] In this embodiment, the parameters of mechanical beating are optimized. Under the premise of selecting enzyme digestion method A, the optimal beating frequency is selected at 37°C. The results are judged by combining the complete digestion time with the cell survival rate. The results are shown in Table 1.

[0048] Table 1 Mechanical beating parameter optimization

[0049] It can be seen that the best dissociation method of the present invention is enzyme digestion method A (collagenase type I 2mg / ml, DNase I10U / ml) combined with mechanical treatment method II (beating treatment, 180 times / min, 37°C). This combination achieves rapid and effective digestion of skin tissue within 15 minutes, while ensuring a cell survival rate of >90%. Further treatment with hyaluronidase and red blood cell lysate, combined with 30-micron filtration and centrifugation multiple purification steps, successfully prepared a high-purity, high-viability single-cell suspension. This method is fast and gentle, greatly improving the efficiency of single-cell preparation, and providing reliable technical support for subsequent flow cytometry analysis, cell sorting, and high-throughput sequencing research.

[0050] Test Example 3

[0051] In this example, we used the AcanCreERT2; Rosa26-LSL-tdTomato fluorescent tracing mouse constructed by us to prepare single cell suspension. This type of mouse can specifically mark the dermal sheath cells of the skin, which is manifested by the expression of tdTomato orange-red fluorescence in the dermal sheath cells of the hair follicle bulb after Tamoxifen induction. The tissue digestion method used is the aforementioned combination 4, and it is processed by the above-mentioned cell purification process. The flow cytometry sorting equipment used is the SONY SH800 sorting system, and the chip nozzle size is 70μm. The flow analysis results are shown in Figure 4 : The overall situation of cells and fragments can be seen in All Events; after the above optimization Efficient tissue dissociation and debris removal The technology has substantially improved the sorting efficiency. The "peak" of tdTomato fluorescent cells has been clearly visible in Events (B), and the Parent ratio has reached 7.91%, which has laid a solid foundation for efficient sorting and analysis.

[0052] The newly obtained highly purified fluorescent tracer cells (skin dermal sheath cells) were sorted by flow cytometry, and the results of fluorescence microscopy were as follows Figure 5 After 72 hours of culture in DMEM / F12 (1:1) + 10% FBS, the adhesion of the cells was observed. Figure 6 , the cells are growing well.

[0053] The above embodiments are limited to several preferred implementations of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for rapidly digesting mouse skin tissue into single cells, characterized in that: The following steps are involved: 1) Pretreatment: Mouse skin tissue was used as the object, hair was removed, and then cut into small pieces; 2) Digestion: Add enzymatic solution containing type I collagenase to the above small pieces and perform mechanical constant temperature beating treatment at the same time. The digestion time is ≤15min.

2. A method for rapidly digesting mouse skin tissue into single cells according to claim 1, characterized in that: The mice are adult mice, the skin is back skin, and skin tissue with a diameter of 0.5-1.5 cm is obtained.

3. The method for rapidly digesting mouse skin tissue into single cells according to claim 1, characterized in that: The enzymatic solution is prepared by adding type I collagenase and DNase I into Hanks buffer; The concentration of type I collagenase in the enzymatic solution is 1-3 mg / ml, and the activity of DNase I is 5-15 U / ml.

4. A method for rapidly digesting mouse skin tissue into single cells according to claim 3, characterized in that: The ratio of the amount of the enzymatic hydrolysate to the diameter of the skin tissue is 1-10: 1 ml / cm.

5. The method for rapidly digesting mouse skin tissue into single cells according to claim 1, characterized in that: The mechanical constant temperature beating treatment includes: constant temperature beating treatment, using a sterile transparent plastic bag, 35-40°C constant temperature reciprocating beating, and a frequency of 180-360 times / min; beating using a beating homogenizer.

6. The method for rapidly digesting mouse skin tissue into single cells according to claim 1, characterized in that: After digestion, cell purification is also required: after digestion, hyaluronidase is added to prevent cell clumping during centrifugation, and red blood cell lysis buffer is used to remove potential residual red blood cells, and then a cell filter is used to remove large impurities to obtain tissue sample digestion solution; The tissue sample digestion solution was centrifuged to remove the supernatant containing particle debris, and then resuspended and centrifuged with DMEM medium to remove tissue debris. Finally, the cells were resuspended with an appropriate amount of complete medium containing serum and observed under a microscope, while the activity of the digestive enzyme was terminated.

7. The method for rapidly digesting mouse skin tissue into single cells according to claim 6, characterized in that: The amount of hyaluronidase added is 0.1-1 mg / ml; the centrifugation parameters are 1000-3000 rpm, 2-4 min.

8. The method for rapidly digesting mouse skin tissue into single cells according to claim 6, characterized in that: After the cell purification, the method further includes detecting the cell viability: counting the living cells of the single cell suspension obtained by digestion, and calculating the cell viability by trypan blue staining.

9. Use of the method according to any one of claims 1 to 8 in flow cytometry analysis, cell sorting or high-throughput sequencing.

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