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原代細(xì)胞分離技術(shù)
實驗步驟
Experimental steps
1. 懸浮細(xì)胞的分離方法
1. Separation method of suspension cells
1) 將血液、羊水、胸水或腹水等懸液直接轉(zhuǎn)至離心管中，1000rpm/分鐘離心5分鐘。
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，離心沉淀用無鈣、鎂的PBS清洗后1000rpm/分鐘離心5分鐘。此步重復(fù)兩次。
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) 用培養(yǎng)基重懸，調(diào)整適當(dāng)細(xì)胞濃度后分瓶培養(yǎng)。
3) The cells were resuspended with medium and cultured in bottles after adjusting the appropriate cell concentration.
4) 如選用懸液中某種細(xì)胞，可采用離心后的細(xì)胞分層液收獲目的細(xì)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. 實體組織材料的分離方法
2. Separation method of solid tissue materials
1）機(jī)械分散法
1) Mechanical dispersion method
a. 纖維成分很少的腦組織，部分胚胎組織，用剪刀剪碎至1mm3的組織塊。
a. Brain tissue with little fiber composition and part of embryonic tissue were cut to 1mm3 tissue block with scissors.
b. 用PBS清洗兩次后
b. After cleaning twice with PBS
①用吸管吹打，分散組織細(xì)胞。
① Blow with a straw to disperse tissue cells.
②或?qū)⒁殉浞旨羲榉稚⒌慕M織放在注射器內(nèi)（用九號針），使細(xì)胞通過針頭壓出。
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或在不銹鋼紗網(wǎng)內(nèi)用鈍物（注射器鈍端）壓擠使細(xì)胞從網(wǎng)孔中壓擠出。
③ Or in stainless steel gauze with blunt object (blunt end of syringe) to squeeze cells from mesh.
c. 收集細(xì)胞轉(zhuǎn)入離心管中，1000rpm/分鐘離心5分鐘。
c. The cells were collected and transferred into a centrifuge tube and centrifuged at 1000 rpm / min for 5 minutes.
d. 去上清，加入含血清的培養(yǎng)基，重懸后移至培養(yǎng)瓶中培養(yǎng)。
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）消化分離法
2) Digestion separation method
酶消化分離法（過夜冷消化法）
Enzymatic digestion and separation (overnight cold digestion)
a. 細(xì)胞間質(zhì)較少的軟組織，如肝、腎、甲狀腺、羊膜、胚胎組織、上皮組織等，用Hanks液清洗組織三次，剪成碎塊大小為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組織。
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，搖勻后放4℃過夜。
c. Add 0.25% trypsin, shake well and put it at 4 ℃ overnight.
d. 次日再用Hanks液洗滌，棄去上清，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養(yǎng)基吹打分散，細(xì)胞計數(shù)，按適當(dāng)?shù)臐舛确制颗囵B(yǎng)。
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. 把組織塊剪碎，呈1mm3大小的組織塊。
a. The tissue block was cut into pieces, and the tissue mass was 1mm3 in size.
b. 將碎組織塊在平皿中用無鈣鎂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（胰蛋白酶或膠原酶或EDTA）于37℃水浴中作用適當(dāng)時間（中間可輕搖1~2次），若組織塊膨松呈絮狀可終止，若變化不大可更換一次消化液，繼續(xù)消化直至膨松絮狀為止。
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. 棄去上清，加入含有鈣、鎂離子的培養(yǎng)基中止消化反應(yīng)，并洗滌2-3次后，加入完全培養(yǎng)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，使細(xì)胞充分散開后用紗網(wǎng)過濾后分瓶培養(yǎng)。
e. The cells were blown with a pipette, and then the cells were filtered with gauze and cultured in bottles.