Detailed
1.Primary human hepatocyte, islet cells application
Primary human hepatocyte refers to hepatocytes cultured or frozen immediately after being removed from liver tissue. It's a "gold standard" cell model that can be widely used in basic research and drug development, including liver research, hepatitis virus research and drugs, metabolism and toxicity studies. CFDA clarified in the "Guidelines of the Technical Research of Non-clinical Pharmacokinetics of Drugs" that primary hepatocyte is an important cell model for the evaluation of macromolecule’s in vitro and traditional Chinese medicine extraction technique in vitro. At present, organoid/spheroid, liver chip and other technologies developed based on primary hepatocytes and liver non-parenchymal cells are the trend of new drug development and evaluation in the future. At the same time, primary hepatocytes, as the main players of liver function, can be used for safety and effective cell therapy and bioartificial liver, including "life bridge" of orthotopic liver transplantation, treatment of acute liver failure, treatment of liver metabolic genetic diseases, etc. Primary hepatocytes have many technical limitations including large batch differences, difficulty in separation and freezing and short culture tie, which severely limit the application of human primary hepatocytes.
Human pancreatic islets refer to endocrine cell cluster in pancreatic organs that play a role in regulating changes in blood glucose and mainly include four types of cells, alpha cells, beta cells, delta cells and PP cells. Islet PP cells account for the smallest proportion of islet cells (about 1%) secrete pancreatic polypeptide; δ cells account for 6%~15%, secrete somatostatin; α cells account for 24%~40%, secrete glucagon; Beta cells account for the largest proportion, 60% to 80%, and secrete insulin. Glucagon and insulin are the two most important endocrine hormones that regulate the changes in human blood glucose: glucagon can increase the glucose content in human blood to ensure that the body produces enough heat to maintain function; Insulin, on the contrary, can decrease the glucose content in human blood. The concentration promotes the transport of glucose in blood to the liver and muscle to store synthetic glycogen. Isolation and preservation of pancreatic islet cells can be used in basic scientific research, including drug toxicology research, endocrine research, diabetes mechanism research, diabetes treatment research, etc. More importantly, combined with 3D bioprinting technology or pancreatic islet group reconstruction technology, the final application to clinically cure type 1 diabetes.
2. Source
At present, primary human hepatocyte/islet cells are mainly obtained from clinical donation materials including corpse donation, surgical resection samples, etc. These clinical samples have the most original physiological and disease correlation, and have important basic research and clinical treatment value. Liver tissue materials are mainly divided into the following three categories: 1) Adult donor liver: Under normal circumstances, the liver donated clinically for transplantation has certain circumstances that cannot be used for liver transplantation with a certain probability, including but not limited to organs being too long in vitro 1. The recipient's condition deteriorated or died early, and the operation failed; 2) Split liver transplantation: due to the large size of the donor liver, the liver needs to be surgically trimmed to fit a smaller (child) body, which will result in a larger volume Liver waste; 3) Liver tissues of other types of diseases, such as hemangioma, biliary atresia, etc., require liver or liver tissue removal. The islet material mainly includes:1) Adult pancreas donors: clinically, transplantation of donor pancreas directly into patients with type 1 diabetes carries out a very small scale, and islet transplantation surgery that separates and purifies pancreatic islets from the pancreas and transplants them into the liver of patients The advantages of small side effects, minimally invasive, safe and effective are more favored by medical workers. However, the team that is subject to domestic islet isolation and purification technology is seriously inadequate, and most of the pancreas donors in the clinic are not yet available for diabetes treatment; 2) Pancreas in patients with acute pancreatitis: unsatisfactory drug treatment, recurrent acute pancreatitis Patients can only choose to surgically remove the pancreas.
The above donated materials for liver and pancreas are generally used for teaching, scientific research, or as a medical waste for harmless treatment with the informed consent of the patient's family and ethical requirements. Due to the lack of specialized separation and preservation technologies in hospitals, including large-scale separation, efficient cryopreservation and recovery technologies, most hospitals can only do simple material extraction or direct harmless treatment. Therefore, these valuable human genetic resources of clinical liver tissue and pancreas materials need to be reasonably and effectively preserved and utilized to meet the growing demand for liver cells and islet cells in basic research and clinical treatment in my country.
3 Necessity of cryopreservation of human primary hepatocytes and islet cells
The necessity of primary human hepatocyte cryopreservation mainly includes: 1) To provide research materials and cell sources for the treatment of acute liver failure, there are 1 million new cases of acute liver failure in China every year, based on primary hepatocyte cell therapy, bioartificial New methods such as liver and treatment products can solve the current major clinical needs, and have huge clinical transformation prospects; 2) Human resources are turned into "trash" into "treasure", which is used by hospitals for simple medical materials and harmless treatment Through our company's technology accumulation in the field of primary hepatocytes, hepatocytes can be separated and frozen on a large scale, and a living cell bank can be established to realize the transformation of human liver tissue into "treasure" to meet clinical treatment and scientific research needs.3) It is convenient for long-term storage and long-distance transportation. The cells can be stored for a long time through liquid nitrogen. According to the actual needs of medical workers or scientific researchers, long-distance transport through dry ice or liquid nitrogen; 4) Provide the best for the development of new drugs in China The test model shows that the drug CYP metabolic enzymes vary greatly due to different races. The best cell model for drug metabolism for Chinese people is primary hepatocytes with Chinese genetic background. (Remdesivir) for the treatment of new coronavirus, the possible reason is that the metabolism and clearance of redoxivir in the Chinese are different from that of the Americans; 6) Breaking the technical blockage, the cryopreservation and resuscitation of primary liver cells are extremely important As a bottleneck, China has been blocked by the technology of developed countries in Europe and America for a long time, which has led to long-term dependence on imports of key raw materials for biomedicine research and development.
The necessity of islet cell cryopreservation mainly includes:
1) Improve the effective use of donor materials. At present, there are insufficient teams in China to master the technology of human islet cell separation. The clinically rich donor pancreas is in sharp contrast with the huge number of patients with type 1 diabetes The imbalance of supply and demand has caused great waste; 2) The urgent need for diabetes treatment, the number of patients with type I diabetes in China is about 6-10 million. Islet transplantation is a safe method to cure type I diabetes, and the grafts survive on average Up to 9.8 years, to avoid irreversible damage to the solid organs caused by insulin injection, improve the quality of life of type I diabetes and reduce its medical burden;3) Improve cell yield and pancreatic islet freezing viability. At present, the isolation and cryopreservation of pancreatic islets are limited to the overall separation and cryopreservation of pancreatic islet cell masses. A single cell is considered to be non-functional, which reduces the cell yield; On the one hand, the islet is a cell mass. The cells inside the cell mass are poorly frozen and recovered due to the infiltration of the cryopreservation fluid and uneven heating; we enzymatically digest the purified islet cell mass into islet single cells to carry out the cell Cryopreservation and resuscitation, after recovery, islet single cells are reconstructed in vitro to form endocrine function cell clusters (islets), which improves the cell yield and islet frozen survival; And cryopreservation technology can well solve the problem of the space-time distance between type 1 diabetes patients and pancreas donors, and at the same time solve the need for secondary or even multiple transplantation when the transplantation effect is poor.
Relevant articles published
Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes.
Zhou M, Huang Y, Cheng Z, Zhao F, Li J, Zhi X, Tian X, Sun W, Hu K.
J Virol Methods. 2014 Oct;207:29-37. doi: 10.1016/j.jviromet.2014.06.015. Epub 2014 Jun 28.
PMID: 24977316
Primary human hepatocytes are considered the ideal cellular model for in-vitro studies of liver-specific pathology, such as hepatitis B virus (HBV) infection. ...Moreover, this optimized model was susceptible to HBV infection and could be used to screen entry inhibi …