CN112831460A

CN112831460A - Method for preparing single cell suspension from lung puncture tissue

Info

Publication number: CN112831460A
Application number: CN202010727575.9A
Authority: CN
Inventors: 姚梦琳; 张立; 田攀文; 蔡绪雨; 汪周峰; 李为民
Original assignee: West China Hospital of Sichuan University
Current assignee: West China Hospital of Sichuan University
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2021-05-25

Abstract

本发明公开了一种新的从肺部穿刺组织中制备单细胞悬液的方法，属于细胞分离与培养领域。本发明的方法包括如下步骤：1)将肺部穿刺组织置于冰上剪碎至颗粒状；2)加入终浓度为0.2mg/ml的IV型胶原酶，37±0.3摄氏度下消化10‑15min；3)使用孔径40μm的细胞过滤网过滤，收集滤过液；4)加入红细胞裂解液重悬细胞。本发明的方法相比传统方法，可以有效提高细胞得率，提高细胞活性。The invention discloses a new method for preparing single cell suspension from lung puncture tissue, which belongs to the field of cell separation and culture. The method of the present invention includes the following steps: 1) placing the lung puncture tissue on ice and shredding it into particles; 2) adding type IV collagenase with a final concentration of 0.2 mg/ml, and digesting it at 37±0.3 degrees Celsius for 10-15 min ; 3) Use a cell filter with a pore size of 40 μm to filter, and collect the filtrate; 4) Add red blood cell lysate to resuspend the cells. Compared with the traditional method, the method of the present invention can effectively improve the cell yield and improve the cell activity.

Description

Method for preparing single cell suspension from lung punctured tissue

Technical Field

The invention belongs to the field of cell separation and culture.

Background

The preparation of single cell suspension from solid tissue is an important basic technology for establishing cell models and researching cell physiological and biochemical mechanisms. The preparation method is different for different tissues.

Loosening hepatic tissue cells, mainly preparing single cell suspension by mechanical grinding method (Korean duckweed, etc., analyzing and comparing hepatic tissue single cell suspension of liver disease patient with lymphocyte subpopulation in peripheral blood, examining medicine, 2005, 20 th volume, 5 th phase); the muscle tissue is compact, and is matched with mechanical grinding and is matched with a plurality of digestive enzymes to act simultaneously, so that a better preparation effect can be realized (Zhang Yuan, etc., compared with the mechanical-enzyme digestion method for preparing rat diaphragm tissue single cell suspension, Chinese tissue engineering research, 24 th volume at the end of 2020, No. 8).

The lung puncture tissue is taken out by puncturing the puncture needle from the outside of the body into the lung, has the characteristics of less number of puncture tissues, more fibrous tissues, easy adhesion of red blood cells to the tissues in the puncture process and the like, and is difficult to prepare single cell suspension from the lung puncture tissue, so that higher cell number and higher activity cannot be ensured.

The routine preparation of single cell suspensions from lung puncture samples comprises the following steps: (1) cutting the punctured tissue; (2) performing enzymolysis on collagenase IV for 30 min; (3) filtering by 70 mu m; (4) and (6) washing. The conventional method prepares single cell suspension from each puncture sample (weight 5-10mg), the number of single cells is usually less than 10000, and the activity is less than 50%. The method leads the research work of taking single cell suspension as the material to have the difficulties of material shortage, low fault tolerance rate and the like, and limits the development of related research work.

Currently, a high yield, high activity method for preparing single cell suspensions is lacking.

Disclosure of Invention

The invention aims to solve the problems that: an improved method for preparing single cell suspensions from punctured tissue of the lung to increase the number and activity of cells in the single cell suspension is provided.

The technical scheme of the invention is as follows:

a method of preparing a single cell suspension from punctured tissue of the lung comprising the steps of:

1) placing the lung puncture tissue on ice and cutting into pieces;

2) adding IV type collagenase with final concentration of 0.2 + -0.05 mg/ml, and digesting at 37 + -0.3 deg.C for 10-15 min;

3) filtering with a cell filter screen with the aperture of 40 mu m, collecting filtrate, and centrifuging to enrich cells;

4) adding PBS containing BSA to resuspend the cells, and centrifuging to enrich the cells;

in step 4), the BSA-containing PBS is obtained by adding 4 + -0.05 mg BSA per 100ml PBS.

As the method, after the step 3), adding erythrocyte lysate to resuspend the cells if the filtrate contains erythrocytes, standing for 5 +/-1 min on ice, and centrifuging to enrich the cells; if no red blood cells are present, the process proceeds directly to step 4).

As described above, the final concentration of collagenase type IV in step 2) was 0.2 mg/ml.

As described above, in step 4), BSA-containing PBS was added in an amount of 4mg BSA per 100ml PBS.

As in the previous method, step 3) and/or step 4), the centrifugation conditions were 400g at 4 ℃ for 6 min.

The invention has the following beneficial effects:

1) the cell number in the prepared cell suspension is large, and is improved by 30 times compared with the conventional method;

2) the cell activity of the prepared cell suspension is high, and is improved to more than 95 percent compared with the cell activity of less than 50 percent of the cell activity of the cell suspension prepared by the conventional method.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed Description

Example 1: method for preparing single cell suspension from lung punctured tissue

The method comprises the following steps:

1) the collection tube with the punctured tissue specimen was centrifuged (300g, 2min, 4 ℃), 950ul of tissue preservative solution (HBSS) was discarded, approximately 100ul of HBSS was left, and the lung punctured tissue sample tube (sample weight 5-10mg) was placed on ice.

2) The tissue was minced, 850ul of HBSS and 50. mu.l of collagenase type IV (2mg/ml) were added, and digested in an incubator at 37 ℃ for 10-15 min.

3) And after digestion is finished, transferring the digestive juice to a centrifuge tube with the volume 4 times that of the digestive juice to obtain HBSS, slightly blowing and beating the HBSS into the centrifuge tube with 15ml by using a 1ml pipette gun for 5-10 times by adhering to the wall, filtering the digestive juice to the centrifuge tube with 50ml through a cell filter screen with the size of 40 mu m (HBSS is rinsed in advance), flushing the filter screen with Hank's Balanced Salt Solution (HBSS) for 3-4ml, flushing the collecting tube with 1ml HBSS, collecting flushing liquid and filtering, collecting residual collecting liquid which does not drip at the bottom of the filter screen by using a pipette tip with the size of 1ml to reduce cell loss, ensuring the total volume to be about 10ml, and centrifuging (400g, 4 ℃ and 6min) to.

4) If the collected liquid contains blood cells, adding 0.5-1ml of erythrocyte lysate to resuspend the cells, standing on ice for 6min, centrifuging (400g, 4 ℃, 6min), and discarding the supernatant; if no blood cells are present, the next step is performed directly.

5)1ml of 0.04% BSA + PBS was added (formulation: 4mg BSA +100ml PBS, the same applies below), transferring the cell into a 1.5ml low adsorption EP tube, centrifuging (400g, 4 ℃, 6min), discarding the supernatant, and reserving 100-200 mul to obtain the single cell suspension to be prepared.

6) The appropriate volume of 0.04% BSA + PBS was counted and 5ul mixed fluorochrome +5ul cell suspension (cell counting plate with luna instrument) was used to determine final cell concentration and activity.

The advantageous effects of the present invention are further illustrated in the form of experimental examples.

Experimental example 1 comparison of the method of the present invention and the conventional preparation method

1. Material selection

The lung puncture samples were obtained from the university of Sichuan Wash Hospital for a total of 217 cases. A single cell suspension was prepared using the conventional preparation method for 126 of the samples, and the single cell suspension was prepared using the method of the present invention for 91 of the samples.

2. Method of producing a composite material

2.1 operating procedure of the conventional preparation method

(1) The collection tube with the punctured tissue specimen was centrifuged (300g, 2min, 4 ℃), 800ul of tissue preservative solution (HBSS) was discarded, about 200ul of HBSS was left, and the lung punctured tissue sample tube (sample weight 5-10mg) was placed on ice.

(2) Tissue (5-10mg) was minced in 200ul, 700ul HBSS added, and finally 100ul of 2mg/ml collagenase type IV, digested in a rotary incubator in an oven at 37 ℃ for 30 min.

(3) The cell suspension was filtered through a 70 μm filter screen (HBSS pre-rinse) and transferred to a 50ml centrifuge tube. Then 1ml of HBSS was aspirated to wash the tissue digestion tube, filtered through a filter, centrifuged (500g, 5min, 4 ℃), and the supernatant was discarded.

(4)5ml of 0.04% BSA + PBS was resuspended in the sample, centrifuged (500g, 5min, 4 ℃ C.), and the supernatant discarded.

(5)1ml of 0.04% BSA + PBS resuspended sample, transferred to a 1.5ml low adsorption EP tube, and the cell count determined the concentration volume; centrifuging (500g, 5min, 4 ℃), discarding the supernatant, and reserving 100-200 mul to obtain the single cell suspension to be prepared.

(6) The appropriate volume of 0.04% BSA + PBS was counted and 5ul mixed fluorochrome +5ul cell suspension (cell counting plate with luna instrument) was used to determine final cell concentration and activity.

2.2 methods of the invention

The same as in example 1.

3. Results

The number of cells in the suspension prepared by the conventional preparation method was 4.14X 10⁴±0.7×10⁴The cell activity is 47.9 +/-4 percent; the number of cells in the suspension prepared by the method of the present invention was 1.32X 10⁶±8×10⁴Cell viability 96.6. + -. 2% per ml.

In conclusion, the method of the invention can successfully prepare the single cell suspension with high cell content and high cell activity from the lung punctured tissue.

Claims

1. a method for preparing single cell suspension from lung puncture tissue, is characterized in that, comprises the steps:

1) Put the lung puncture tissue on ice and chop it up;

2) Add collagenase IV with a final concentration of 0.2±0.05mg/ml, and digest at 37±0.3 degrees Celsius for 10-15min;

3) Use a cell filter with a pore size of 40 μm to filter, collect the filtrate, and centrifuge to enrich the cells;

4) Add BSA-containing PBS to resuspend the cells, and centrifuge to enrich the cells;

In step 4), the BSA-containing PBS is obtained by adding 4±0.05 mg of BSA to every 100 ml of PBS.

2. method as claimed in claim 1 is characterized in that: after step 3), if filtrate has red blood cells, then add red blood cell lysate to resuspend cells, leave standstill on ice for 5 ± 1min, and centrifuge to enrich cells; If there are no red blood cells, go directly to step 4).

3. The method of claim 1, wherein the final concentration of type IV collagenase in step 2) is 0.2 mg/ml.

4. method as claimed in claim 1 is characterized in that: in step 4), the PBS containing BSA is obtained by adding 4mg BSA on the basis of every 100ml PBS.

5. method as claimed in claim 1 is characterized in that: in step 3) and/or step 4), centrifugal condition is 400g centrifugation 6min under 4 degrees Celsius.