Transport Mode
Liquid Nitrogen Transportation
1、Adsorptive Liquid Nitrogen Transportation ,No free liquid nitrogen ,White vapor emission indicates normal operation.
2、Temperature monitoring devices should closely monitor the temperature inside the tank during transportation. If any abnormalities are detected, you can copy the PDF and Excel data from the temperature recorder. (One end of the temperature monitoring device can be unplugged to reveal a USB connector. Simply insert it into a computer to copy the data. If you encounter any difficulties, you can contact the sales staff and technical support for assistance.)
3、Alloy Combination Lock Designed to ensure no third-party access to the LN₂ tank or cell samples during transportation from dispatch to receipt, thereby safeguarding cell integrity.
4、GPS Tracker Enables real-time tracking of cell transportation routes to prevent loss.
5、Liquid Nitrogen Tank, temperature monitoring device, alloy combination lock, and GPS tracker are returnable components. Please store them properly and avoid damage; failure to comply will result in blacklisting.
Reference Citation
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Scope of Application
Liver Kupffer cells are the most important macrophage population in the liver, accounting for 80-90% of the sedentary macrophages in the whole body, and their use in scientific research is extensive and involves the following main directions:
1. Research on the mechanism of liver fibrosis and cirrhosis
Kupffer cells activate hepatic stellate cells (HSCs) by secreting pro-inflammatory factors (e.g., TNF-α, IL-1β, TGF-β) to drive the fibrotic process.
M1/M2 phenotypic regulation: the M1 type promotes fibrosis (secretion of pro-inflammatory and pro-fibrotic factors), while the M2 type is involved in fibrotic regression (through secretion of anti-inflammatory factors and degradation of extracellular matrix).
Metabolic reprogramming: to study the effects of its metabolic pathways, such as glycolysis and fatty acid oxidation, on polarization, and to provide a basis for targeted metabolic antifibrotic therapy.
2. Research on liver cancer microenvironment and tumor progression
Kupffer cells have dual roles in hepatocellular carcinoma occurrence and development:
Anti-tumor function: inhibit tumor growth by phagocytosis of tumor cells and secretion of cytotoxic factors; decreased number of Kupffer cells in hepatocellular carcinoma tissues correlates with tumor progression.
Pro-tumor mechanism: activated Kupffer cells remodel the tumor microenvironment and support invasion and metastasis of hepatocellular carcinoma cells by secreting pro-angiogenic factors such as VEGF.
4. Drug development and toxicity assessment
Anti-fibrotic drug screening: assess the modulatory effects of drugs on M1/M2 polarization, collagen secretion using immortalized Kupffer cell lines (e.g. rat/human primary cells).
Nanomedicine targeting: study the clearance mechanism of nanoparticles by their scavenger receptors (e.g. Stabilin-1/2) and optimize the drug delivery system to bypass non-specific uptake.
5. Metabolic diseases and cross-organ interactions
Lipid metabolism studies: Kupffer cells regulate hepatic lipid deposition through pathways such as STAT3, which is closely related to the pathogenesis of non-alcoholic fatty liver disease (NAFLD).
Role of the gut-liver axis: processing of metabolites such as LPS of intestinal origin is involved in the influence of intestinal flora on liver disease.
Summary
The scientific uses of Kupffer cells cover a wide range of areas including liver disease mechanisms (fibrosis, hepatocellular carcinoma), immune regulation, drug development and metabolic studies, and their functional heterogeneity and metabolic plasticity provide new targets for disease treatment. For example, targeting M1/M2 polarization or metabolic pathways may become a new strategy for anti-fibrosis or anti-tumor. Experimental models (e.g., primary cell culture, gene-edited animals) and single-cell histology technologies will further advance the mechanism resolution.
Due to biannual updates of our product instruction manual, the following procedures are for reference only.
The most recent version included with the product shall prevail.
II 试剂与材料
Ø 肝Kupffer细胞（Cat# LV- Kup001/2/3/4）
Ø 肝Kupffer细胞培养基（Cat# LV-KuPM001）
Ø 培养板（TC-treated）
Ø 无菌15ml离心管
Ø 一次性移液管
Ø 37℃/5%CO2培养箱
Ø 移液枪
Ø 恒温水浴锅（37℃预热）
Ø 冷冻水平离心机（带水平转子，可离心15ml离心管）
Ø 生物安全柜
Ø 宽口移液枪头
Ø 75%酒精
重要提示：解冻时，冻存细胞转移必须使用液氮转移；在整个复苏实验过程中必须全程使用宽口枪头。
III 细胞的复苏与铺板
1. 肝Kupffer细胞培养基放入37℃恒温水浴锅中充分预热。
2. 将冻存的肝Kupffer细胞从冷藏位置通过液氮迅速转移至37℃恒温水浴锅中。将冻存管尽可能多的浸入37℃水中，顺时针水平摇动，但必须确保冻存管管盖保持在水面以上。
3. 解冻冻存管约90~120s，至冻存管中只有少量碎冰漂浮即可。
4. 用75%酒精消毒冻存管，并将其转移到生物安全柜。
5. 用宽口枪头将细胞重悬（轻轻吹打2次），并转移至含10ml预热培养基的15ml离心管中（注意：冻存管与枪头上残留细胞，可用1ml复苏培养基润洗冻存管与枪头）。
6. 将细胞逐滴加入预热培养基中，最后轻微上下颠倒3次混匀。
7. 600g，4℃离心5min，去上清并用培养基重悬。
8. 沉淀的细胞用肝Kupffer细胞培养基重悬并定容至4ml，用台盼蓝排除法测定细胞的活力和细胞总量。
9. 将细胞按3×104cells/cm2接种至TC处理培养板中。摇匀，在37℃/5%CO2培养箱中培养，24h后换液。
IV 细胞培养与传代
1. 细胞融合度达到80%时可进行传代。
2. 提前将培养基、PBS、胰酶放入37℃水浴锅内预热，用75%酒精擦拭后再放入超净台内。
3. 吸除旧培养液，加少量PBS润洗细胞，加入适量胰酶，使加入的胰酶能盖住细胞，37℃孵育，约2~3min后显微镜下观察。
4. 待贴壁细胞间间隙变大、细胞趋于圆形但还未漂起时弃去胰酶，加入新鲜培养基，晃动细胞板，终止胰酶作用，用吸管小心吹打贴壁的细胞，制成细胞悬液（控制吹打的力度，避免产生大量的气泡）。
5. 600g，室温离心5min，去上清并用肝Kupffer细胞培养基重悬。
6. 将细胞悬液按3×104cells/cm2接种到新的TC处理培养瓶/板内，置37℃/5%CO2培养箱中培养，隔天观察贴壁生长情况。
