As mentioned above, the main concern in HT is the lack of adequate sources for viable human hepatocytes. Current sources of liver tissue are mainly adult organs considered unsuitable for whole LT, often of marginal quality. The HCTU collaborates closely with the National Transplant Organization (NTO) to recover donated organs that have been deemed unsuitable for transplantation.

Most of the organs for hepatocyte isolation have been discarded for transplantation due to steatosis greater than 40%-50%[7,27], prolonged ischaemia time, traumatic damage to the graft[47], capsular tear[47,54] blood group incompatibility[9] and vascular or biliary lesions. Occasionally, fetal hepatocytes have been used for infusion[43].

New sources for hepatocyte isolation include liver tissue resulting from hepatic reductions, split liver grafts and segment 4 after a split-liver graft for two patients, grafts from donors after circulatory death, and donors with severe arteriosclerosis[48,49,54].

Steatotic donor livers are becoming more common with the increasing incidence of obesity in our population. For this reason, at the beginning of the program, the HCTU used to accept most of the organs discarded for transplantation, regardless of the percentage of steatosis, in order to not waste any possible source of hepatocytes[16,17,22,27,30,31]. However, based on the poor results, we decided to refuse steatotic liver grafts discarded for transplantation as a source for HT.

Most of the criteria to accept a liver for cellular isolation are similar to those required for LT donation. Appropriate liver donors for HT are all those grafts rejected for LT, with no age limit, that show no signs of sepsis or history of neoplasia, with negative serology for hepatitis C and B, HIV, HTLV, and syphilis, family consent for research purposes, steatosis less than 50%, no hepatic illnesses (cirrhosis, cholestasis, or haemophilia), and a cold ischaemia time less than 12 h when possible.

Neonatal donor: Our experience shows that this is not enough and new sources of human liver tissue for hepatocyte isolation need to be explored. An interesting alternative to adult liver is the use of unsuitable neonatal livers for LT as a potential source of differentiated hepatic cells which, to our knowledge, has been poorly explored to date[3,56,57].

In the HCTU neonatal donors are considered a potential source of hepatocytes[29]. In 2010, a program for neonatal liver donation was set up in our center as a new source for hepatocytes after a national approval by the NTO and the ethic committee. These livers are not usually suitable for LT due to low weight, immature liver function and the technical difficulties related to the size of the vessels and the biliary tree (Figure 2). However, a high yield of good-quality hepatocytes can be isolated from neonatal liver tissue[29].

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Figure 2 Liver from a neonatal donor to be used for hepatocyte isolation.

In neonatal donors, brain death certification is very difficult to verify. Despite the brain stem test in newborns, toddlers, and children being similar to that in adults, there are some differences as to the duration of the observation period and also with the instrumental tests performed, which make it much more difficult to diagnose. This inconvenience led us to consider another type of donation, such as donation after circulatory death. According to the Maastrich classification, types III and IV donors are an important source of grafts for transplantation, especially type III, in which asystole happens after the controlled withdrawal of all the therapeutic measures[58,59].

The newborn period includes the first 28 d of life. Our inclusion criteria for organ donation were patients whose pathologies did not respond to medical and/or surgical treatment and with life incompatibility: congenital anomalies, intracranial intraventricular hemorrhage grades III-IV, congenital disorders of the central nervous system with no activity in the electroencephalography (EEG), surgical pathology not amenable to treatment and incompatible with life, anencephalic, cranioencephalic trauma, premature newborns with pathologies that do not respond to therapeutic maneuvers, surgery patients with postoperative cerebral anoxia[58]. Patients with HIV-1 and 2, HBV, HCV, and sepsis with multiorgan failure were excluded.

Our laboratory works from a standardized protocol for hepatocyte isolation from surgical hepatic biopsies, which has been optimized and adapted for large scale isolations from organs unfit for whole-organ transplantation or from liver fragments from hepatectomies[10,13,15]. At the early stage of isolation, we perfused the entire organ resulting in an incomplete digestion of the hepatic tissue[22].

Another difficulty was to avoid digestion of the vessel cannulated to infuse the collagenase before the digestion of the parenchyma has been achieved. This spurred a modification of the perfusion system, and we divided the hepatic parenchyma into left and right lobes, performing a separate perfusion of each lobe[22,25] (Figure 3). Similar situations were described by Mitry et al[55], suggesting the use of one donor for three recipients, proposing segment IV obtained from a split, as a cellular isolation source.

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Figure 3 Optimization of the process of hepatocyte isolation. When livers weighing 1.5-3.5 kg are available, a better recovery was achieved by dividing the hepatic parenchyma (0.5-1 kg) and perfusing each lobe.

In general, during the disgregation process of the liver or its lobes, a better output of the lightest samples (one lobe) was noticeable. However, splitting the liver prolonged the procedure affecting the viability and the functionality of the hepatocytes isolated due to longer cold ischaemia time. To prevent this, we performed the digestion of both lobes simultaneously in two different laminar flow hoods.

Splitting the liver created new problems in the process for hepatocytes isolation. By performing an ex situ division of the hepatic graft in right lobe and left lobe, we had to suture all the arterial, portal and suprahepatic branches in the cut surface to avoid leakage of the collagenase, leading to a tedious process due to the large amount of ligatures and stitches used, increasing graft preparation time for more than 3 h. To prevent this, we applied TachoSil^®, a hemostatic sealant used during hepatectomies to stop small bleeding points and small bile leaks in the cut surface[25] (Figure 4A).

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Figure 4 Split liver sutured the arterial, portal and suprahepatic branches in the cut surface. A: The application TachoSil^®, a hemostatic sealant, avoids leakage of the collagenase; B: Cannulation of the hepatic veins allows using bigger calibre cannulas increasing the flow of the perfusates.

Before using the TachoSil^®, we tried different biological glues, regularly used in surgical practice to seal blood vessels from the hepatic transection margin. However, the results showed that these glues were not able to tolerate collagenase pressure from the infusion pump, leading to leakage from multiple openings. As a result, we observed an incomplete digestion, which conditioned a decrease in cellular output. Moreover, we verified the presence of glue residues in the cell solution, being impossible to separate them from the cells, which disabled them for their infusion. The use of TachoSil^® in human hepatocytes isolation provides us great advantages: avoids suture and ligation of arterial and venous branches with reduction in preparation time, as well as cold ischemia time of the graft, crucial factor of cellular viability and performance. Moreover, by pointing out the leak of the recirculating solution, it enables an ideal collagenase perfusion of the graft, vital step in the hepatic parenchyma digestion and, therefore, in the hepatocytes isolation[25]. In contrast with other haemostatic agents and sealants, TachoSil^® has not been detected in the cell suspension infusion.

Apart from the cannulation of the portal vein, another modification was to cannulate the hepatic veins, which allowed using bigger calibre cannulas increasing the flow of the collagenase. This also contributed to a better digestion of segment 7 and 8 due to its proximity to the hepatic veins. With all these modifications, a better digestion of the liver parenchyma was achieved, improving the efficiency and the viability of the isolated hepatocytes (Figure 4B).

In the case of neonatal livers, a super rapid technique was used to procure them due to the reduced size of these organs. The hepatic veins, portal vein or umbilical vein within the umbilical cord were identified and were therefore cannulated according to vessel size. This cannula was then connected to the tissue preservation solution (Celsior^® solution; Genzyme, Madrid, Spain), and liver perfusion started. The liver was placed into two or three sterile bags and transported to the laboratory while being maintained in ice-cold Celsior^® solution until cell isolation[24,29]. The digestion process in these livers is usually faster (10-15 min) and uniform, with a better efficiency and viability when comparing to adult livers[29].

A standardized collagenase perfusion technique is used to digest liver tissue to obtain human hepatocytes[3,6,10,16,22,44,60]. Briefly, major liver vessels are cannulated and perfused using a peristaltic pump with buffered salt solution containing chelating agent EGTA, which removes calcium and disrupts desmosomes; the liver is subsequently perfused with collagenase solution to digest the extracellular matrix[3,10,24,59]. Initially, enzymatic perfusion is performed following cannulation of the hepatic artery and the portal vein from the donated liver tissue (Figure 5A). When feasible, we also cannulate the biliary duct[22]. During the perfusion procedure, the liver and solutions are kept at 37 °C to allow optimal enzyme activity. After digestion, the liver tissue is disrupted, and the suspended hepatocytes are purified by filtration and low-speed centrifugation (Figure 5B-F). The average hepatocyte yield is 3 × 10⁶-20 × 10⁶ hepatocytes per gram of liver tissue[6,13,14,22] and the conditions for tissue isolation influence the procedure results[61].

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Figure 5 Hepatocyte isolation and transplantation process. A: Perfusion of the liver at 37 °C; B: Liver after digestion by collagenase perfusion; C: Purification of hepatocytes by low-speed centrifugation; D and E: Hepatocyte suspension is washed twice and pelleted by low-speed centrifugation; F: Freshly isolated hepatocyte suspension; G: Cell thawing by immersing the bags in a fixed-temperature bath at 37 °C; H: Hepatocyte suspension in infusion medium ready to be transplanted; I: Infusion of hepatocytes to the receptor patient.

Different concerns have arised over the years in the digestion process. The first concern was to reach 37 °C in a uniform manner throughout the liver, especially in the thickest part of the right lobe corresponding to segments 7 and 8. This resulted in an incomplete digestion of the liver parenchyma on the right lobe while the left lobe was already digested. To resolve this problem, the temperature of the bath was increased up to 42 °C with the liver submerge and contained with three organ bags, which helped to preserve the sterility of the procedure and also to reach the target temperature for the collagenase to work properly.

Based on our studies with liver biopsies from surgical resections, the protocol was optimized and updated to perform hepatocytes isolation on a large scale using those livers discarded for transplantation or bigger portions of liver specimens from hepatectomies[10,13,14].

The experience gained with the results of the first hepatocyte isolation from steatotic grafts, helped us to optimize the process of hepatocyte isolation and cultivation culture from whole livers (1.5-3.5 kg) or complete lobes (0.5-1 kg) unused for transplantation (Figure 3).

We performed 8 isolations from livers discarded for transplantation because of the severe steatosis[16,17,22] (Table 1). Hepatic steatosis has become a common problem in our populations with an increased incidence of obesity. It affects about 25% of liver transplant donors and most of the steatotic donor liver are rejected for organ transplantation[27,30,62,63].

Organs with advanced steatosis (≥ 40%) showed lower cell yields during isolation (viable hepatocytes obtained/g of tissue processed). Our experience has allowed us to demonstrate that the hepatocytes isolated from these livers are usually of low cell viability and poor function. Steatotic hepatocytes do not tolerate cryopreservation well, leading to greater cell loss, impaired metabolic function and the chances of a successful engraftment are lower, compared to cell from nonsteatotic livers[13,14,17,22,44]. According to Sagias et al[63] and based on the results, we decided to decline livers rejected for transplantation with the background of steatosis as a source for HT.

Livers from elderly donors showed tendencies of reduced viability (Table 1). In general, the disintegration process of the liver in this case, gives lower yields than those obtained through surgical biopsies of fatty liver tissue, although in some cases higher yields were observed. For this reason, livers form elderly donors are also discarded as a source of hepatocytes.

Initially, the HCTU recovered 13 donor organs deemed unsuitable for LT, but fit for HT. The hepatocytes were isolated from donors with arteriosclerosis, doubtful viral serology, donors after circulatory death, split liver grafts, fibrotic and steatotic livers[22]. All grafts were irrigated with Celsior^® preservation solution during the procurement, because in our experience, its use produces a better cell viability in the human hepatocytes isolation process[14,15], compared with other preservation fluids[15].

Of the 13 donor livers (8 women, 5 men), the isolation was performed in 3 split lobes, 2 whole organs, and in the remaining 8 donor livers, the organ was divided into 2 lobes, left and right lobes to improve the digestion. The percentage of steatosis was determined by pathology studies. The cold ischaemia time remained under 4 h in all cases and the resulting cell viability was between 70% and 89% with a yield of 1 × 10⁶-17 × 10⁶ cells/g (Table 2)[22].

In general, the disintegration process of the whole liver or split lobes gives lower yields than those obtained through surgical biopsies from fatty liver tissue, although we must also highlight higher yields from livers with lower weight.

As a result of this work, we created a biobank of cryopreserved hepatocytes functionally characterized and ready to be used for HT. The first HT by HCTU was performed in our hospital on May 2008 in a girl with urea cycle defect.

The clinical outcome after HT depends very much on the quality of the hepatocytes used, which depends to a large extent on the nature of the tissue utilized for cell isolation, donor factors such as age, and cell harvesting procedures[16-18,20,24,30,31,64].

In addition, from the surgical removal of tissue to the final obtaining of cell suspensions, hepatocytes are subjected to a number of factors [i.e., warm ischemia and hypoxia during surgical liver resection, cold ischemia during transportation, the cell isolation procedure, the perfusion liquid (Celsior^®/University of Wisconsin (UW) solution] that can influence their viability and functionality[14,17,31]. For instance, it seems to be a negative correlation between hepatocyte viability and the duration of both warm and cold ischemia[60].

The trypan blue assay is quick and easy to perform and is the most frequently used test to evaluate cell number and viability[20,31]. However, this assay cannot detect early apoptotic cells or evaluate the metabolic function of the cell. In general, viability greater than 50%-60% is considered the minimum for clinical transplantation[3,20,59]. Simple viability measurements are not conclusive enough to predict the long-term HT outcome, and a correlation between in vitro hepatocyte viability and the graft function after transplantation is lacking[20,59]. In our experience, the hepatocytes with a initial viability below 70% generally decrease 20% of viability after cryopreservation/thawing. This does not happen in those hepatocytes isolated from neonatal donors[29].

Hepatocytes should not only be highly viable but also functionally active to support those functions lacking in the recipient liver. Therefore, other assessment criteria are needed for a better estimation of the quality of hepatocytes and to provide an objective basis for decision making.

Because we encounter frequently donor livers of poor quality, fast and sensitive procedures to evaluate the functional performance of hepatocyte preparation have been sit up in order to determine their suitability for HT[18,59]. To this end, reliable assays have been developed to assess the functional capacity of cells in the course of the isolation procedure or shortly after thawing. Key hepatic functions, such as the synthesis of plasmatic proteins, urea synthesis or ammonia removal, and drug-metabolizing capacity, have been assessed for this purpose[3,18,59,65]. In fact, a rapid quality assessment of freshly isolated hepatocytes could help to estimate their potential susceptibility to freezing/thawing-induced cell damage and the convenience of banking a particular hepatocyte preparation.

We measured cell yield and functionality through metabonomic studies of phase I and II biotransformation enzymes, ureogenesis capabilities, and total number of cells[11,12] (Figure 1).

In the HCTU we have established specific criteria for functional quality control of the hepatocytes (fresh or cryopreserved) so that we can establish their suitability for cell transplantation in a reasonable time frame (1 h). The activity of 5 major P450 cytochrome isoenzymes (CYP1A2, CYP2A6, CYP3A4, CYP2C9 and CYP2E1) is determined along with phase II conjugation enzymes [UDP, glucuronosyltranferase (UGT) and sulfotransferease] by incubation with a specific substrate cocktail recently developed in our laboratory, high performance liquid chromatography/mass spectrometry analysis[6,18-20]. In the future, we hope to use metabonomic studies and establish a useful number of cellular metabolites to characterize the quality of hepatocytes.

Innovative methods to improve cell quality have been a focal point of research. Hepatocytes perform a wide range of functions that can be individually tested after isolation. We aimed to assess the functionality of hepatocyte preparations with a view to promote customized cell preparation for each receptor to fit the metabolic defect of the recipient[6,17,24].

Neonatal hepatocytes: In our experience, neonatal hepatocytes showed better post-thawing recovery compared with adult hepatocytes, as shown by the viability values that did not differ significantly from freshly isolated cells, a higher expression of adhesion molecules (b1-integrin, b-catenin, and E-cadherin), better attachment efficiency, cell survival, and a lower number of apoptotic cells. The metabolic performance of thawed hepatocytes has been assessed by ureogenesis and drug metabolizing capability (cytochrome P450 and UGT enzymes). CYP2A6, CYP2C9, CYP2E1, and CYP3A4 activities were found in all cell preparations, while CYP1A2, CYP2B6, CYP2C19, and CYP2D6 activities were detected only in hepatocytes from a few neonatal donors. The expression of UGT1A1 and UGT1A9 (transcripts and protein) was detected in all hepatocyte preparations, while activity was measured only in some preparations, probably due to lack of maturity of the enzymes. However, isoforms UGT1A6 and UGT2B7 showed considerable activity in all preparations[27]. Compared to adult liver, the hepatocyte isolation procedure in neonatal livers also provides thawed cell suspensions with a higher proportion of hepatic progenitor cells (EpCAM+ staining), which could also participate in regeneration of liver parenchyma after transplantation[8,27]. These results could imply important advantages of neonatal hepatocytes as a source of high-quality cells to improve human HT applicability[29].

The time lag between the isolation of hepatocytes and their use causes heavy losses of many resources due to the lack of a method for conservation during prolonged periods of time. Cryopreserved cells have the advantages that they are constantly available and that extensive quality testing can be performed, whereas rapid assays are necessary for fresh cells in order to determine suitability for transplantation[4,6,22,25,65,66]. Another advantage of cryopreservation is that it allows a more effective quarantining of cells against bacterial or fungal contamination as compared with using fresh cells. For this reason, only cryopreserved cells are used in our Unit. This provides a biobank of cryopreserved cells which allows us to perform semi-elective HT with previous microbiological and functional assessment[6,7,22].

However, cryopreservation has detrimental effects on the viability and metabolic function of hepatocytes, and therefore, on thawed cells, they are often not suitable for clinical use[3,6,24,66]. Thawed cell viability and function are related to the nature of the liver tissue from which hepatocytes were isolated, including factors such as the donor’s age or condition, liver steatosis, and the duration of cold and warm ischemia times involved in procuring the liver[66]. However, hepatocytes isolated from fetal or neonatal livers can be cryopreserved without significant loss of viability or function[3,29].

Another major factor is the speed of freezing, if the velocity is too slow, the hepatocyte can suffer dehydration, but if it is too fast, the intracellular liquid fails to exit and freezes. The consequence of this process is the formation of crystals that, together with fluctuations in cellular volume, cause damage to the cellular membrane and rupture of the cell, causing cellular necrosis[67]. In recent studies, the different steps involved in the hepatocyte cryopreservation and thawing process were systematically investigated and included cell density, addition of cryoprotectants, freezing temperature program, and thawing method[26,68,69].

However, no consensus has been reached on the use of fresh or cryopreserved hepatocytes, and some centers prefer transplanting fresh hepatocytes because of better viability and function as compared to cryopreserved cells[6,7,41,43,45,46]. Despite these limitations, cryopreserved hepatocytes are often used for clinical transplantation in some patients and yield a good metabolic effect[6,7,16,46].

Another approach involves developing methods for the cold storage of hepatocytes. This has been recently demonstrated to be possible for up to two weeks using new organ preservation solutions[61].

Our protocol to cryopreserve hepatocytes has been submitted to substantial modifications over time. Cryopreservation is accomplished by controlling the freezing rate in a solution containing a permeable cytoprotectant dimethyl sulfoxide (DMSO). According to current protocols, hepatocytes are frozen in a mixture of UW solution with DMSO (10%-15%). The suspension is frozen in 250 mL bags containing only 50 mL in each with a cell density of 20 million cells/ml using a programmed temperature reduction biological freezer (CM- 2000, Metal Carbides) until reaching -140 °C. The cells are then stored in liquid nitrogen at -196 °C. For each bag prepared, 4 criovials are frozen in order to characterize the functionality of the cells before thawing the bags for infusion.

Cell thawing is performed by immersing the bags in a fixed-temperature bath at 37 °C. The cryopreservation solution is then diluted using the appropriate medium, centrifuged at 4 °C for 5 min, and the supernatant is discarded. The cellular pellet is resuspended in an appropriate volume of the medium for infusion into the patient, and cellular viability is assessed[22,26,31] (Figures 1 and 5G).

From the 13 isolations performed at the HCTU[22], cell viability, which reached 78%-89% following isolation, significantly decreased in all cases after prolonged cryopreservation storage and thawing (Table 2)[20,31].

At present, there are three generally used routes for cell transplantation: portal vein, splenic vein, and intraperitoneal duct. Injection through the portal vein (Figure 5I) should be reserved for correcting inborn metabolic errors, while the splenic artery should be considered for patients with a fibrotic liver.

Cells are most often delivered by the catheterization of the portal or inferior mesenteric vein by laparoscopic surgery and infusion[29]. The spleen can be used as an alternative transplantation site, especially in patients with liver cirrhosis. The spleen can be accessed by direct injection into the splenic artery through a catheter inserted through the femoral artery for implantation in the spleen. Cells are manually infused. Thus, during the cell infusion, a Doppler portal vein ultrasound and intraportal pressure and a repeated Doppler ultrasound after cell infusion are essential to exclude thrombosis formation.

The hepatocyte mass required to cure a liver-based metabolic disease is expected to vary according to the specific metabolic deficiency. Transplanted hepatocytes are ABO compatible with the recipient.

The hypothetical aim is to perform repeated cell infusions in order to provide an approximate total number of cells representing 5%-10% of the theoretical liver mass. This also depends on the use of fresh vs cryopreserved cells[3,9,29,30,47]. It is still unclear what constitutes the maximum number of liver cells that can be infused each time and how many infusions can be performed in all. As the average adult liver contains 2.8 × 10¹¹ hepatocytes, no more than 1%-2% of liver mass can be transplanted in each single-cell infusion[2,70]. Nevertheless, repeated infusions can be undertaken at time intervals by monitoring portal pressure. In children, cells (up to hepatocytes 10⁸/kg of body weight) are infused slowly (5-10 mL/kg per hour) based on the original considerations of Fox et al[9] and Horslen et al[50] for the purpose of replacing about 5% of the liver cell mass. Furthermore, cell dose and infusion rate are adjusted to patient size and clinical situation.