1.肝臟細胞分類(lèi)
肝臟是人體最大腺體,包含兩大類(lèi)細胞,分別是肝實(shí)質(zhì)細胞(PC)和肝非實(shí)質(zhì)細胞(NPC)。
肝臟實(shí)質(zhì)細胞包括了肝細胞和膽管細胞。肝細胞約占所有肝細胞的70%,并負責大部分肝代謝功能。
肝非實(shí)質(zhì)細胞約占總肝細胞的30%。肝非實(shí)質(zhì)細胞包括巨噬細胞(Kupffer cells, KC)比例約35%、肝竇內皮細胞(Liver endothelial cells, LEC)比例約45%和肝成纖維細胞(Liver fibroblasts)。也就是說(shuō)巨噬細胞和肝竇內皮細胞一起占據了80%。
2.肝臟內不同細胞分離富集
2.1肝組織灌洗:一方面可以去除肝細胞的紅細胞,并通過(guò)膠原蛋白酶灌注提高肝組織的利用率,另一方面可以收集肝臟血液中的白細胞等其他免疫細胞。肝臟灌洗替代的方式可以將肝臟組織剪成2-4 毫米的小塊,加入適量的酶并在最佳溫度(通常為 37°C)下孵育適當的時(shí)間,間歇混合。最終將組織消化后通過(guò)篩網(wǎng)過(guò)濾細胞懸浮液。
2.2根據肝臟中各細胞的物理特性對肝內細胞進(jìn)行分離和提純。
例如由于粒徑的差異,肝細胞的粒徑普遍在100um左右,而KC、LEC、成纖維細胞的粒徑普遍小于100um,因此可以40um或70um的細胞篩網(wǎng)篩去除不需要的肝細胞,也可以使用50g離心5min將肝細胞(會(huì )沉淀)和肝臟其他細胞(繼續懸?。┓蛛x開(kāi)來(lái)。由于各細胞的密度差異,死細胞或細胞殘渣由于密度小,在層析液中移動(dòng)慢,因此使用不同密度梯度的層析液可以有效地分離死細胞和肝臟內各成分的活細胞。
2.3根據肝臟中各細胞的黏附速度進(jìn)行分離
肝臟中KC與其他非實(shí)質(zhì)細胞相比黏附速度很快,因此將肝臟非實(shí)質(zhì)細胞提取放置在細胞培養箱中約30min,KC就會(huì )貼壁,而其他類(lèi)型的細胞不會(huì )貼壁,此時(shí)吸走細胞培養基,留在培養瓶底的大多數為KC。反復操作可利于收集更多KC。
2.4根據肝臟細胞中各細胞的黏附能力進(jìn)行分離
肝臟中成纖維細胞的黏附能力明顯強于肝臟中其他細胞。因此如果細胞懸液中僅有成纖維細胞和目標細胞,例如LEC或KC等??梢允褂靡让付虝r(shí)間消化后立即收集細胞,反復多次操作利于提高目標細胞的比例。
2.5根據肝臟細胞中各細胞的生長(cháng)速度輔助提純
肝臟中成纖維細胞的生長(cháng)能力很強,如果樣本分離不純使得目標細胞中摻雜了成纖維細胞,可以使用含1%FBS培養基限制成纖維細胞的生長(cháng),當細胞密度大于50%后,再通過(guò)胰酶快速消化的方法純化目標細胞。
3.肝臟內不同細胞分離的器械、試劑或耗材
3.1 器械:準備鑷子、刀片、留置針、50ml注射器/恒流泵(強烈建議用恒流泵)、外科線(xiàn)、外科剪、轉運盒、冰塊。
3.2 試劑:DMEM培養基、RPMI培養基、Hanks液、PBS、血清、肝細胞培養基(或購買(mǎi)地塞米松、胰島素、白蛋白、谷氨酰胺等)、EDTA/肝素、雙抗/三抗、膠原蛋白酶Ⅳ型、層析液Percoll、乙醇。
3.3 耗材:培養皿、培養瓶、細胞篩網(wǎng)、移液管、移液槍、50ml和15ml離心管。
4.肝臟灌注,密度梯度離心方法Intrahepatic macrophages isolation
Primary mouse macrophages were isolated from the mouse liver. Briefly, mouse livers were first perfused in situ with Ca2+ and Mg2+-free Hank’s balanced salt solution (HBSS) containing 0.5 mM EGTA through the portal vein and then again with HBSS containing 0.05% collagenase IV. The digested livers were torn open and then gently separated with forceps until they were in suspension. The cell suspensions were filtered with a 70-μm cell filter, and the hepatocytes were removed by differential centrifugation (50×g, 5 min; 72×g, 5 min). To obtain nonparenchymal cells, the supernatant was collected and centrifuged further (300×g, 5 min; 650×g, 7 min). The pellets from both centrifugation steps were pooled and resuspended in DMEM containing 2% fetal bovine serum (FBS). The cell suspension was transferred to a 25%/50% Percoll gradient and centrifuged at 1200×g for 30 min without braking. The macrophage fraction was enriched in the interface between the 25% and 50% Percoll layers. Macrophages were purified by using a mouse F4/80 positive selection EasySep kit (EasySep™ Mouse F4/80 Positive Selection Kit; 100-0659) in accordance with the manufacturer’s instructions.
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