实验步骤

Experimental steps

1. 悬浮细胞的分离方法

1. Separation method of suspension cells

1) 将血液、羊水、胸水或腹水等悬液直接转至离心管中，1000rpm/分钟离心5分钟。

1) The suspension of blood, amniotic fluid, pleural fluid or ascites was directly transferred into a centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes.

2) 去掉上清，离心沉淀用无钙、镁的PBS清洗后1000rpm/分钟离心5分钟。此步重复两次。

2) The supernatant was removed, centrifuged and precipitated with PBS without calcium or magnesium, and centrifuged at 1000 rpm / min for 5 minutes. Repeat this step twice.

3) 用培养基重悬，调整适当细胞浓度后分瓶培养。

3) The cells were resuspended with medium and cultured in bottles after adjusting the appropriate cell concentration.

4) 如选用悬液中某种细胞，可采用离心后的细胞分层液收获目的细胞。

4) If a certain cell is selected in the suspension, the centrifuged cell layering solution can be used to harvest the target cells.

2. 实体组织材料的分离方法

2. Separation method of solid tissue materials

1）机械分散法

1) Mechanical dispersion method

a. 纤维成分很少的脑组织，部分胚胎组织，用剪刀剪碎至1mm3的组织块。

a. Brain tissue with little fiber composition and part of embryonic tissue were cut to 1mm3 tissue block with scissors.

b. 用PBS清洗两次后

b. After cleaning twice with PBS

①用吸管吹打，分散组织细胞。

① Blow with a straw to disperse tissue cells.

②或将已充分剪碎分散的组织放在注射器内（用九号针），使细胞通过针头压出。

② Or put the fully cut and scattered tissue into the syringe (with No. 9 needle) to press out the cells through the needle.

③或在不锈钢纱网内用钝物（注射器钝端）压挤使细胞从网孔中压挤出。

③ Or in stainless steel gauze with blunt object (blunt end of syringe) to squeeze cells from mesh.

c. 收集细胞转入离心管中，1000rpm/分钟离心5分钟。

c. The cells were collected and transferred into a centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes.

d. 去上清，加入含血清的培养基，重悬后移至培养瓶中培养。

d. The supernatant was removed, and the culture medium containing serum was added. After suspension, it was transferred to the culture bottle for culture.

2）消化分离法

2) Digestion separation method

酶消化分离法（过夜冷消化法）

Enzymatic digestion and separation (overnight cold digestion)

a. 细胞间质较少的软组织，如肝、肾、甲状腺、羊膜、胚胎组织、上皮组织等，用Hanks液清洗组织三次，剪成碎块大小为4毫米左右。

a. Soft tissue with less intercellular substance, such as liver, kidney, thyroid, amnion, embryonic tissue and epithelial tissue, should be washed with Hanks solution for three times and cut into pieces about 4 mm in size.

b. 再用Hanks液洗2-3次以除去血球和脂肪组织。

b. Wash 2-3 times with Hanks solution to remove blood cells and adipose tissue.

c. 加入0.25%的胰蛋白酶，摇匀后放4℃过夜。

c. Add 0.25% trypsin, shake well and put it at 4 ℃ overnight.

d. 次日再用Hanks液洗涤，弃去上清，共洗2-3次。

d. The next day, wash with Hanks solution, discard the supernatant, and wash 2-3 times.

e. 加入少量含血清的培养基吹打分散，细胞计数，按适当的浓度分瓶培养。

e. Add a small amount of serum containing medium, blow and disperse, count cells, and culture in bottles according to appropriate concentration.

非酶消化法（EDTA消化法）

Non enzymatic digestion (EDTA digestion)

a. 把组织块剪碎，呈1mm3大小的组织块。

a. The tissue block was cut into pieces, and the tissue mass was 1mm3 in size.

b. 将碎组织块在平皿中用无钙镁PBS洗2-3次。

b. Wash the broken tissue block in the plate with PBS without calcium and magnesium for 2-3 times.

c. 加入消化液（胰蛋白酶或胶原酶或EDTA）于37℃水浴中作用适当时间（中间可轻摇1~2次），若组织块膨松呈絮状可终止，若变化不大可更换一次消化液，继续消化直至膨松絮状为止。

c. Add the digestive solution (trypsin or collagenase or EDTA) into the water bath at 37 ℃ for a proper time (gently shaking 1-2 times in the middle). If the tissue block is bulky and flocculent, it can be stopped. If the change is not big, the digestive solution can be replaced once and digestion will continue until it is fluffy.

d. 弃去上清，加入含有钙、镁离子的培养基中止消化反应，并洗涤2-3次后，加入完全培养基。

d. Discard the supernatant, add medium containing calcium and magnesium ions, stop digestion reaction, and wash 2-3 times, then add complete medium.

e. 用吸管吹打，使细胞充分散开后用纱网过滤后分瓶培养。

e. The cells were blown with a pipette, and then the cells were filtered with gauze and cultured in bottles.