Towards a bank of cryopreserved hepatocytes: which cell to freeze?
Article Outline
- 1. Why are hepatocytes required?
- 2. Can hepatocytes be cryopreserved?
- 3. Why use progenitor or stem cells rather than mature hepatocytes?
- 4. Cryopreservation of small hepatocytes
- References
- Copyright
1. Why are hepatocytes required?
The results of studies conducted with animal models indicate that hepatocytes transplanted into the liver or spleen survive, function, and participate in the normal regenerative process [1], [2]. Recent clinical research suggests that hepatocyte transplantation may be useful as a bridge for patients to whole organ transplantation, and that it may provide metabolic support during liver failure. Hepatocyte transplantation may also be able to take the place of whole organ transplantation in the treatment of certain metabolic liver diseases [1], [3]. In recent years, techniques have been developed to isolate, culture, and cryopreserve human hepatocytes on a large scale. However the use of mature hepatocytes (MH) raises several problems, among them: maintaining the stability of metabolic function following cell isolation, difficulties in cryopreservation, immunogenicity, and inadequate response to growth factors. MH can be maintained in culture; however, they cannot be expanded very effectively. Moreover, mature hepatocytes can repopulate the liver only in the event of extensive ongoing liver damage, or an inability of endogenous hepatocytes to proliferate, as both instances would provide a substantial selection advantage for the transplanted cells, as compared with host hepatocytes. These conditions may not always be present in many cases where cell transplantation might be otherwise regarded as a useful form of therapy [4].
2. Can hepatocytes be cryopreserved?
The scarcity of donor livers is a major obstacle to the general application of hepatocytes for cell transplantation, and the development of bioartificial liver assisted devices. Some of these limitations could be overcome by the use of cryopreserved organs [5], [6]. However, the freeze–thaw conditions necessary for successful cryopreservation of hepatocytes have not yet been well defined. The most critical parameters appear to be choice of cryoprotectant; composition of the freezing medium; the cooling and thawing rates; concentration of the cryoprotectant; slow addition and removal of freezing solution; carbogen equilibration during isolation and before cryopreservation; and removal of unvital hepatocytes after thawing [7], [8].
It is difficult to identify which factors are important in determining cell viability following cryopreservation, because of the vast differences among the various methods for cryopreservation that have been documented. Research in organ preservation has led to the development of flush solutions to buffer the harsh molecular conditions that develop during ischemia. These solutions also provide stored organs fit to sustain life after transplantation. However, the methods used thus far in cryopreservation appear to be limited in terms of the duration, preservation, and quality of the organs or cells that can be preserved.
3. Why use progenitor or stem cells rather than mature hepatocytes?
It is generally accepted that the liver contains cells with stem-like properties and that these cells can be activated to proliferate and differentiate into mature hepatic epithelial phenotypes under certain pathophysiologic circumstances [4], [9]. The isolation and enrichment of hepatic progenitor cells by fluorescent activated cell sorting, and the development of a colony-forming assay to characterize these cells, represent two of the important advances in the quest to identify the liver stem cell and how it may be used for effective liver repopulation [4]. In recent years hepatic progenitors were proposed as ideal cells for use in liver cell therapies, due to their ability to expand extensively, differentiate into all mature liver cells, and their relative minimal immunogenicity. These cells are more metabolically stable, may not be dependent on environmental factors for their growth and differentiation, and are easier to grow [9]. In contrast to MH, stem/progenitor cells continue to proliferate long after transplantation, and may not need great ‘selection pressure’ to ultimately repopulate the liver. Moreover, although transplanted MH remain at high levels and for a long period of time in rodent livers, this is not the case in humans.
Finally, it has been difficult to introduce foreign genes into hepatocytes in vitro.Therefore, effective gene therapy might be more readily achieved with liver stem/progenitor cells. It was also recently suggested that progenitor cells may be more cryopreservable, with better chances of reconstituting liver tissue when transplanted [4]. The cumulative effect of these benefits would include expansion of the donor pool, extension of the safe preservation period, and an increase in metabolic effectiveness of the cells.
4. Cryopreservation of small hepatocytes
In the current issue of the Journal, Ikeda et al. suggest a method for cryopreservation of small hepatocytes (SHs) [10]. The authors have previously described how a single SH could clonally differentiate and form a large colony within 10 days [11]. It was suggested that SHs are a type of a ‘committed progenitor cell’. These cells were also shown to proliferate into MH and to interact with other non-parenchymal cells. SHs were able to form three-dimensional structures, and to survive for 5 months while maintaining albumin secretion and bile formation. SH colonies were surrounded by a thin layer of extracellular matrix (ECM), which may provide both protection and necessary growth factors [11].
The aim of the study was to show that these cells can be cryopreserved for a long period. SHs survived at −80 °C for more than 6 months, and after thawing, they proliferated while maintaining hepatic differentiated functions. The results showed that 60% of cryopreserved colonies attached themselves to petri dishes and proliferated, and that albumin production increased. The length of the cryopreservation period did not influence the attachment rate of the SH colonies. Most of these colonies maintained a monolayer. Isolated cells cryopreserved without culture did not grow, while SHs that attached to the dish continued to proliferate. The amount of albumin secreted into culture medium increased as SH colonies grew. Cells after a year of cryopreservation expressed transferrin, fibrinogen, and carbamoyl phosphate synthetase. TO, an enzyme expressed by mature hepatocytes, was also detected in the cells 35 days following thawing. When SHs were cultured with a small number of non-parenchymal cells, they rapidly proliferated without maturation.
Several explanations are given for these results, including the three dimensional structure of the colonies that may protect SHs from physical or physiological damage. Another possible explanation is a cell-cell contact that may be important to maintaining a stable condition. There is good evidence that preservation times would be extended by the provision of continuous cellular substrate [4]. Thus the effect that was achieved could be due to the fact that SHs create a colony, with less area exposed to the surroundings, in comparison with isolated cells, and to the fact that a thin layer of ECM surrounded the colony – which may provide the required environment for the cells – may be held accountable for the effect achieved.
In the study, no in vivo evidence for the function of SHs is shown. However, in another recent study, transplantation of SHs was more effective than that of MH [12]. One of the most difficult aspects of this type of research is to show the potency of the isolated progenitor cells. Both clonality and metabolic functions of isolated liver stem/progenitor cells needs to be confirmed by in vivo transplantation studies [4]. These studies will need to show multiple cells in clusters that exhibit potency as evidenced by simultaneous expression of markers for hepatocytes and cholangiocytes in the same cells; proliferation and differentiation of these cells into hepatocyte cords and mature bile ducts; and the formation of complete new liver lobules.
In summary, there are many reasons for the extensive effort to use progenitors cells as targets for cryopreservation. The study of Ikeda et al. [10] is one of several that have recently been reported on the isolation and cryopreservation of liver stem/progenitor cells. All these studies indicate that the potential for liver repopulation by cryopreserved transplanted stem cells may become a reality.
References
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PII: S0168-8278(02)00131-9
© 2002 European Association for the Study of the Liver. Published by Elsevier Inc. All rights reserved.
