METHOD FOR EXTRACTING EPIDERMAL CELLS

Abstract

The present disclosure discloses a method for extracting epidermal cells. The method includes soaking skin tissues in a digestive juice, and obtaining an epidermal cell suspension through alternate use of a high-temperature digestion condition and a low-temperature digestion condition. The method can be used for clinical research or treatment of burns, scalds, pigmentation and hypopigmentation, ulcers, aesthetic dermatology and the like. Through adjusting the digestion conditions of the skin tissues, the method separate the epidermis from the dermis within a relatively short time, and have the advantages of extracting epidermal cells in large amount, high survival rate and high activity. The extracted epidermal cells are applicable to various clinical indications.

Claims

1. A method for extracting epidermal cells, comprising: soaking skin tissues in a digestive juice, and obtaining an epidermal cell suspension through alternate use of a high-temperature digestion condition and a low-temperature digestion condition, the method specifically comprising the following steps: 1) taking out the skin tissues in a sterile environment, putting the skin tissues into a culture dish, and washing the skin tissues with normal saline; 2) soaking epidermal tissues in the digestive juice to perform digestion, wherein a digestion environment in the digestive juice involves alternation of the high-temperature digestion condition and the low-temperature digestion condition or the low-temperature digestion condition and the high-temperature digestion condition; 3) after digestion, separating epidermis from dermis; and 4) resuspending the epidermal tissues with normal saline to obtain the epidermal cell suspension; wherein the method is capable of obtaining the epidermal cell suspension within 4 h, and in the cell suspension, a cell extraction amount is greater than 110.sup.7 cells/10 cm.sup.2, and the cell survival rate is higher than 85%.

2. The method for extracting epidermal cells according to claim 1, wherein step 2) specifically comprises: soaking the skin tissues in the digestive juice, performing digestion at 25 C. to 40 C. for 0.1 h to 2 h and then at 0 C. to 10 C. for 0.1 h to 2 h, and then performing digestion under the above conditions alternately; or soaking the skin tissues in the digestive juice, and performing digestion at 0 C. to 10 C. for 0.1 h to 8 h and then at 25 C. to 40 C. for 0.1 h to 8 h.

3. The method for extracting epidermal cells according to claim 1, wherein a total time range for the high-temperature digestion condition is 0.1 h to 8 h, and a total time range for the low-temperature digestion condition is 0.1 h to 8 h.

4. The method for extracting epidermal cells according to claim 3, wherein the skin tissues are derived from a human or an animal.

5. The method for extracting epidermal cells according to claim 1, wherein the digestive juice is any one, two or three of collagenase, dispase, a TrypLE cell dissociation agent, trypsin, ethylenediamine tetraacetic acid, and an Accutase cell dissociation agent.

6. The method for extracting epidermal cells according to claim 5, wherein a concentration range of the collagenase is 50 U/mL to 1000 U/mL, a concentration range of the dispase is 0.01 U/mL to 10 U/mL, a concentration range of the TrypLE cell dissociation agent is 0.01 to 10, a concentration range of the trypsin is 0.01% to 10%, a concentration range of the ethylenediamine tetraacetic acid is 0.01% to 10%, and a concentration range of the Accutase cell dissociation agent is 0.01 to 10.

7. The method for extracting epidermal cells according to claim 1, wherein the epidermal cells are used for clinical research or treatment of burns, scalds, pigmentation and hypopigmentation, ulcers, aesthetic dermatology and the like.

Description

DETAILED DESCRIPTION

[0024] The technical solution of the present disclosure will be further explained by specific examples below:

Example 1

[0025] This example is specifically implemented as follows: [0026] 1. Epidermal tissues from a human were taken out in a sterile environment, put into a culture dish, and washed with normal saline. [0027] 2. The epidermal tissues were equally divided into 4 groups, and the surface area of each group was 10 cm.sup.2: [0028] group 1: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at room temperature for 2 h; [0029] group 2: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/ml dispase, digested at room temperature for 2 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0030] group 3: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, and digested at 10 C. for 4 h; and [0031] group 4: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, and digested at room temperature for 4 h. [0032] 3. After digestion, epidermis was separated from dermis. [0033] 4. After the epidermal tissues were resuspended with normal saline, counting was performed, and a cell survival rate was determined. [0034] 5. The cells were inoculated and cultured for 24 h, and a growth activity was determined by an MTT method.

[0035] Implementation results are shown below:

TABLE-US-00001 Cell Cell Cell extraction survival activ- Group amount rate ity Group 1: digested at 10 C. for 2 h, 2.56 10.sup.7 cells 88.66% 0.91 and digested at room temperature for 2 h Group 2: digested at room 2.93 10.sup.7 cells 86.72% 0.90 temperature for 2 h, and digested at 10 C. for 2 h Group 3: digested at 10 C. for 4 h 1.33 10.sup.6 cells 74.91% 0.82 Group 4: digested at room 4.33 10.sup.6 cells 70.91% 0.71 temperature for 4 h

[0036] The above results of this example show that within the same digestion time, compared with group 3 (constant-temperature digestion at low temperature) and group 4 (constant-temperature digestion at high temperature), group 1 (digestion at alternate low temperature and high temperature) and group 2 (digestion at alternate high temperature and low temperature) significantly increase the cell extraction amount, survival rate and activity. Compared with constant-temperature digestion at low temperature and constant-temperature digestion at high temperature, the digestion at alternate high temperature and low temperature increases the cell extraction amount by 4 to 20 times (from less than 5.010.sup.6 cells/10 cm.sup.2 to more than 2.510.sup.7 cells/10 cm.sup.2), increases the cell survival rate by 1.13 to 1.21 times (from less than 75% to more than 85%), and increases the cell activity by 1.13 to 1.29 times (from less than 0.85 to more than 0.90). The results of this example indicate that the cell extraction amount, survival rate and activity can be improved by alternately using high temperature and low temperature.

Example 2

[0037] This example is specifically implemented as follows: [0038] 1. Epidermal tissues from a human were taken out in a sterile environment, put into a culture dish, and washed with normal saline. [0039] 2. The epidermal tissues were equally divided into 3 groups, and the surface area of each group was 10 cm.sup.2: [0040] group 1: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at room temperature; [0041] group 2: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, digested at room temperature for 2 h, and then transferred to be subjected to digestion at 10 C.; and [0042] group 3: soaked in a digestive juice composed of 100 U/mL collagenase and 1 U/mL dispase, and digested at 10 C. [0043] 3. At 4, 6, 8, 10 and 12 h of digestion, whether the tissues had been completely digested was observed, and the digestion was stopped after complete digestion.

[0044] Implementation results are shown below:

TABLE-US-00002 Digestion time (h) Condition 4 6 8 10 12 Group 1: digested at Almost Completely / / / 10 C. for 2 h, and then completely digested transferred to be digested subjected to digestion at room temperature Group 2: digested at Completely / / / / room temperature for 2 digested h, and then transferred to be subjected to digestion at 10 C. Group 3: digested at Incompletely Incompletely Incompletely Incompletely Completely 10 C. digested digested digested digested digested

[0045] The results of the above example show that the digestion time of group 1 (digestion at alternate low temperature and high temperature) and group 2 (digestion at alternate high temperature and low temperature) is shorter than that of group 3 (constant-temperature digestion at low temperature). Compared with constant-temperature digestion at low temperature, the digestion at alternate high temperature and low temperature can shorten the digestion time by 2 to 3 times (from more than 10 h to less than 4 h). The results of this example indicate that the extraction time can be shortened by alternately using high temperature and low temperature.

Example 3

[0046] This example is specifically implemented as follows: [0047] 1. Epidermal tissues from the back of a mouse were taken out in a sterile environment, put into a culture dish, and washed with normal saline. [0048] 2. The epidermal tissues were equally divided into 4 groups, and the surface area of each group was 10 cm.sup.2: [0049] group 1: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at 37 C. for 2 h; [0050] group 2: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 37 C. for 2 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0051] group 3: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at 37 C. for 2 h; [0052] group 4: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 37 C. for 2 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0053] group 5: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at 45 C. for 2 h; [0054] group 6: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 45 C. for 2 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0055] 3. After digestion, epidermis was separated from dermis. [0056] 4. After the epidermal tissues were resuspended with normal saline, counting was performed, and a cell survival rate was determined. [0057] 5. The cells were inoculated and cultured for 24 h, and a growth activity was determined by an MTT method.

[0058] Implementation results are shown below:

TABLE-US-00003 Cell Cell Cell extraction survival activ- Group amount rate ity Group 1: digested at 10 C. for 2 h, 3.06 10.sup.7 cells 88.20% 1.32 and digested at 37 C. for 2 h Group 2: digested at 37 C. for 2 h, 3.53 10.sup.7 cells 85.34% 1.02 and digested at 10 C. for 2 h Group 3: digested at 10 C. for 2 2.28 10.sup.6 cells 73.43% 0.83 h, and digested at 37 C. for 2 h Group 4: digested at 37 C. for 2 h, 2.34 10.sup.6 cells 74.26% 0.82 and digested at 10 C. for 2 h Group 5: digested at 10 C. for 2 h, 3.36 10.sup.6 cells 63.30% 0.68 and digested at 45 C. for 2 h Group 6: digested at 45 C. for 2 h, 3.65 10.sup.6 cells 65.31% 0.66 and digested at 10 C. for 2 h

[0059] The results of above example show that within the same digestion time, compared with group 3 (the low temperature being 10 C. and the high temperature being 37 C.), group 4 (the low temperature being 10 C. and the high temperature being 37 C.), group 5 (the low temperature being 10 C. and the high temperature being 45 C.) and group 6 (the low temperature being 10 C. and the high temperature being 45 C.), group 1 (the low temperature being 10 C. and the high temperature being 37 C.) and group 2 (the low temperature being 10 C. and the high temperature being 37 C.) significantly increase the cell extraction amount, survival rate and activity. Compared with the high temperature above 40 C. and the low temperature below 0 C., the condition of the high temperature ranging from 25 C. to 40 C. and the low temperature ranging from 0 C. to 10 C. increases the cell extraction amount by 10 to 15 times (from less than 4.010.sup.6 cells/10 cm.sup.2 to more than 3.010.sup.7 cells/10 cm.sup.2), increases the cell survival rate by 1.13 to 1.31 times (from less than 75% to more than 85%), and increases the cell activity by 1.22 to 1.69 times (from less than 0.85 to more than 1.00). The results of this example indicate that the cell damage can be reduced and the cell extraction amount, survival rate and activity can be improved by controlling the temperature ranges of the high temperature and the low temperature.

Example 4

[0060] This example is specifically implemented as follows: [0061] 1. Epidermal tissues from the back of a mouse were taken out in a sterile environment, put into a culture dish, and washed with normal saline. [0062] 2. The epidermal tissues were equally divided into 6 groups: [0063] group 1: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at 37 C. for 2 h; [0064] group 2: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 37 C. for 2 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0065] group 3: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 2 h, and then transferred to be subjected to digestion at 37 C. for 10 h; [0066] group 4: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 37 C. for 10 h, and then transferred to be subjected to digestion at 10 C. for 2 h; [0067] group 5: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 10 C. for 10 h, and then transferred to be subjected to digestion at 37 C. for 2 h; [0068] group 6: soaked in a digestive juice composed of 1TrypLE cell dissociation agent and 1 U/mL dispase, digested at 37 C. for 2 h, and then transferred to be subjected to digestion at 10 C. for 10 h; [0069] 3. After digestion, epidermis was separated from dermis. [0070] 4. After the epidermal tissues were resuspended with normal saline, counting was performed, and a cell survival rate was determined. [0071] 5. The cells were inoculated and cultured for 24 h, and a growth activity was determined by an MTT method.

[0072] Implementation results are shown below:

TABLE-US-00004 Cell Cell Cell extraction survival activ- Group amount rate ity Group 1: digested at 10 C. for 2 h, 3.23 10.sup.7 cells 85.20% 1.21 and digested at 37 C. for 2 h Group 2: digested at 37 C. for 2 h, 3.66 10.sup.7 cells 86.22% 1.10 and digested at 10 C. for 2 h Group 3: digested at 10 C. for 2 h, 4.36 10.sup.6 cells 66.31% 0.71 and digested at 37 C. for 10 h Group 4: digested at 37 C. for 10 4.22 10.sup.6 cells 63.28% 0.63 h, and digested at 10 C. for 2 h Group 5: digested at 10 C. for 10 3.86 10.sup.6 cells 70.36% 0.83 h, and digested at 37 C. for 2 h Group 6: digested at 37 C. for 2 h, 3.55 10.sup.6 cells 73.67% 0.84 and digested at 10 C. for 10 h

[0073] The results of above example show that within the same digestion time, compared with group 3 (at low temperature for 2 hours and high temperature for 10 hours), group 4 (at low temperature for 2 hours and high temperature for 10 hours), group 5 (at low temperature for 10 hours and high temperature for 2 hours) and group 6 (at low temperature for 10 hours and high temperature for 2 hours), group 1 (at low temperature for 2 h and high temperature for 2 h) and group 2 (at low temperature for 2 h and high temperature for 2 h) significantly increase the cell extraction amount, survival rate and activity. Compared with the high-temperature digestion time of more than 8 h and the low-temperature digestion time of more than 8 h, the condition of the high-temperature digestion time ranging from 0.1 h to 8 h and the low-temperature digestion time ranging from 0.1 h to 8 h increases the cell extraction amount by 10 to 15 times (from less than 4.210.sup.6 cells/10 cm.sup.2 to more than 3.210.sup.7 cells/10 cm.sup.2), increases the cell survival rate by 1.13 to 1.31 times (from less than 75% to more than 85%), and increases the cell activity by 1.22 to 1.69 times (from less than 0.85 to more than 1.10). The results of this example indicate that the cell damage can be reduced and the cell extraction amount, survival rate and activity can be improved by controlling the time ranges of the high-temperature digestion and the low-temperature digestion.