Artiﬁcial Cells Containing Hepatocytes and/or Stem Cells

CHAPTER 9

Artificial Cells Containing Hepatocytesand/or Stem Cells in RegenerativeMedicine

9.1. Introduction

Artificial cells and regenerative medicine

Chapter 8 describes the potential of artificial cells containing islets,cells, genetically engineered cells, microorganisms and stem cells fortreatment of a number of clinical conditions. Many of these clinicalconditions, like diabetes and neurological diseases, require long termtreatment. Chapter 8 also discusses the further development of cellencapsulation technologies needed for long term use. To cater formore immediate use, one area of research in this laboratory focuseson the use of artificial cells in regenerative medicine. We have beenstudying the use of artificial cells containing hepatocytes and/or bonemarrow stem cells for liver regeneration. Fulminant hepatic failure orextensive liver resection for metastatic cancer or severe injury canresult in severe decrease in liver function not compatible with life. Onthe other hand, the liver is an organ that can regenerate itself, giventhe required time and condition. Thus, our aim is to study whetherartificial cells containing hepatocytes and/or bone marrow stem cellscan maintain the experimental animal alive, long enough to allow theliver to regenerate sufficiently for the animal to recover (Fig. 9.1).

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226 Artificial Cells

Fig. 9.1. Upper: Liver failure severe enough to be not compatible with life.This can be caused by acute hepatitis, massive traumatic injury or extensivecancer resection. Liver has the ability to regenerate itself to its original sizeif the patients can survive for a sufficient length of time under suitableconditions. Lower:We studied the use artificial cells with 3 different contentsfor liver regeneration: (1) hepatocytes; (2) hepatocytes plus bonemarrow stemcells; or (3) bone marrow stem cells alone.

Hepatocytes and/or Stem Cells in Regenerative Medicine 227

Design of study

We use artificial cells with three different contents (Fig. 9.1): 1)hepatocytes; 2) hepatocytes plus bone marrow stem cells; and 3) bonemarrow stem cells alone.We first carried out a preliminary feasibility study on the use of

artificial cells containing hepatocytes in the galactosamine inducedfulminant hepatic failure rat model. We then used the Gunn rats,a congenital rat model of hyperbilirubinemia, for more quantitativemeasurement. These studies show the feasibility of using artificialcells to support liver function. Next, we carried out a study todetermine whether artificial cells can prevent immunorejection, forexample, in artificial cells containing rat hepatocytes injected intomice. We also developed an improved method for the encapsulationof hepatocytes in artificial cells. Using this improved method ofpreparing artificial cells containing both hepatocytes and bonemarrow stem cells, we showed that the duration of functions afterimplantation was improved.Furthermore, we studied the use of artificial cells containing only

bone marrow stem cells. The reason for this is that, although theuse artificial cells can help prevent immunorejection without theneed for immunosuppressant, it is difficult to obtain hepatocytesfrom humans. If we use hepatocytes from animals, we can similarlyprevent immunorejection. However, since smaller protein moleculeslike albumin from the hepatocytes can diffuse out of the artificialcells, there is the potential of immunological problems. On theother hand, obtaining bone marrow from humans for use in otherpatients is a routine hematological procedure. Thus, if we canuse artificial cells containing only bone marrow stem cells, thenit would solve many of the potential problems. As describedin this chapter, we show that artificial cells containing bonemarrow stem cells results in the regeneration of the liver andrecovery of the rats which had 90% of their livers surgicallyresected.


9.2. Artificial Cells Containing Hepatocytes

Galactosamine-induced fulminant hepatic failure in rats

This is a preliminary feasibility study (Wong and Chang, 1986).Hepatocytes from 125–135 g Wistar rats were isolated and enclosedin alginate-polylysine-alginate artificial cells as described under“Methods” in Appendix II. Intraperitoneal injection of galactosamine(140mg/100 g body weight) resulted in acute liver failure in theWistar rats of 275–285 g. Forty-eight hours after the galactosamineinjection, the rats in grade II coma were separated into pairs. Onerat in each pair was randomly selected as control and the other fortreatment. A total of 14 rats were used. In the control group, 4mlof alginate artificial cells containing no hepatocytes was injectedintraperitoneally. In the treated group, 4ml of alginate artificial cellscontaining hepatocytes were similarly injected. Each 300 microndiameter artificial cell contained 120± 20 S.D. hepatocytes. The totalnumber of artificial cells was about 62,000 in the 4ml of artificial cellsinjected. Thus, each injection consists of artificial cells containing atotal of about 7.4× 106 hepatocytes. These artificial cells are flexibleand can be easily injected using syringes. The animals were given foodand 5% glucose oral ad libitum. Survival and other parameters weremonitored.Galactosamine-induced fulminant hepatic failure rats in the control

group died 66.1 ± S.D. 18.6 hours after galactosamine injection. Thesurvival time in the group that received artificial cells containinghepatocytes was 117.3 ±D.S. 52.7 h. Paired analysis showed that thisis significantly (P < 0.025) higher than that of the control group. Thetotal number of hepatocytes injected was only a small fraction of thetotal hepatocytes of the rat liver. This preliminary data encouragesus to pursue further studies using artificial cells containing higherconcentrations of hepatocytes to study the effect on survival rates.However, we need to obtain more basic information before pursuingthese studies on survival rates. The galactosamine model is not alwaysreproducible.Thus, we opted to first use a more stable Gunn rat animalmodel where we can carry out more quantitative measurements (Bruni


and Chang, 1989). This would allow us to carry out more meaningfulanalysis on how to improve the artificial cells system.

Gunn rats with severe elevation of bilirubin,hyperbilirubinemia

The homozygous phenotype of Gunn rats (Gunn, 1938), like theirhuman counterpart in Crigler-Najjar syndrome, lacks the liver enzyme,UDP-glucuronyltransferase.This results in severe elevation of bilirubin,termed hyperbilirubinemia, appearing shortly after birth. In humans,death of the infant usually occur in the first year of life. We usedthe Gunn rat model to carry out the following study (Bruni andChang, 1989). Artificial cells containing hepatocytes were prepared(as described in the Methods section of Chapter 8). For this study,2.2ml of a 50% suspension of artificial cells were injected intra-peritoneally into the rats. The total number of viable hepatocytesimplanted in each rat was 15× 106.Figure 9.2 shows the results of the first study on 3.5-month-old

female Gunn rats (Bruni and Chang, 1989). During the 16-day control

Fig. 9.2. One intraperitoneal injection of artificial cells containing a totalof 15 × 106 hepatocytes into Gunn rats. Effects on the lowering of systemicbilirubin (Bruni and Chang, 1989).


period, serum bilirubin increased steadily at a rate of 0.32±0.07mg/dlper day. Twenty days after the intraperitoneal injection of artificialcells containing hepatocytes fromWistar rats, the serum bilirubin leveldecreased from the original 14±1mg/dl to 6.0±1mg/dl. Ninety dayslater, the serum bilirubin was still lower than the control period.This preliminary study was followed by a second study using both

control and treated groups.The effect of artificial cells containing hepatocytes (total hepatocytes

in the artificial cells being 15 × 106) was compared to two controlgroups (Burni andChang, 1989): 1) Gunn rats received control artificialcells containing no hepatocytes; and 2) Gunn rats receiving freehepatocytes (total of 15 × 106). Figure 9.3 shows that the serumbilirubin levels in the Gunn rats which received control artificialcells containing no hepatocytes are significantly higher than thosein the Gunn rats that received artificial cells containing hepatocytes.Figure 9.3 shows that both free hepatocytes and artificial cellscontaining the same number of viable hepatocytes have the same effect

Fig. 9.3. Effect of one intraperitoneal injection of (1) control artificial cellscontaining no hepatocytes; (2) 15×106 free hepatocytes; or (3) artificial cellscontaining a total of 15× 106.


in lowering the serum bilirubin levels. This shows that the methodof preparing artificial cells does not affect the ability of hepatocytesto conjugate bilirubin. The result also shows that free hepatocytesfrom the closely related Sprague-Dawley rats are not rejected by theGunn rats.

Summary

The results up to now show that hepatocytes in artificial cells wheninjected, can carry out their functions. This is shown by the increasein survival time in a galactosamine fulminant hepatic failure ratmodel and the lowering of bilirubin in a hyperbilirubinemic Gunn rat.However, before pursuing further how this can help in the regenerationof the liver, it would be important first, to study whether artificial cellscan protect hepatocytes from immunorejection.

9.3. Immunoisolation

Can artificial cells protect hepatocytes fromimmunorejection?

In order to answer this question, we follow the viability of rathepatocytes implanted into mice either in the free form or inside theartificial cells (Wong and Chang, 1988). However, with implantedartificial cells, the viability of hepatocytes depends on more than onefactor. In addition to immunorejection, there is also fibrous coating andaggregation of artificial cells related to biocompatibility. The result isdecreased viability due to lack of oxygen and nutrients. We opted firstto study only the factor related to immunorejection. This is done byfollowing only those artificial cells that are not aggregated inside thefibrous mass.Hepatocytes were obtained from the livers of male Wistar rats

using the procedure as described in Appendix II. The viability ofthe hepatocytes prepared in this way was 86.0% as measured bytrypan blue exclusion . The hepatocytes were then encapsulated inalginate-polylysine-alginate artificial cells using the method describedin Appendix II. The viability of the hepatocytes using this procedure


was 63.4%. The recipients were 20–22 g male CD-1 Swiss mice.A total of 5 × 106 hepatocytes in either the free form or insideartificial cells were implanted into each mouse intraperitoneally. Atpredetermined intervals, the mice were sacrificed and the peritonealcavity was washed twice with 2.0ml of iced Hanks balanced saltsolution. The viability of the hepatocytes was determined by trypanblue stain exclusion. As mentioned above, only the free artificial cellswere analyzed in this study. The microcapsules were stained for 5 minand then punctured with sharp forceps to release the hepatocyte frominside the artificial cells. The trypan blue stained and unstained cellswere counted under a light microscope and the total number of cellswas measured using a hemocytometer.Figure 9.4 shows the results obtained (Wong and Chang, 1988). The

viability of free rat hepatocytes injected into mice decreased rapidly.

Fig. 9.4. Intraperitoneal injection into each mouse of (1) 15 × 106 rathepatocytes or (2) artificial cells containing a total of 5×106 rat hepatocytes.Trypan blue dye exclusion is the method for analyzing the viability of freehepatocytes and hepatocytes in the artificial cells that are free in the peritonealcavity. (Wong and Chang, 1988).


No viable free hepatocytes were recovered by the 14th day. On theother hand, the viability of the hepatocytes inside the free artificial cellssteadily increased, and by 29 days after implantation, the viability hasincreased from the original 63.4% to more than 90%. The result of thepresent study seems to show that rat hepatocytes in the free form arerejected rapidly after being injected intraperitoneally into mice. Forthe artificial cells that were not aggregated, the rat hepatocytes insidewere protected from rejection after the cells were injected into mice.However, the increase in viability of hepatocytes inside the artificialcells was an unexpected observation. We have therefore carried out aseparate study to find out the reason.

Why do rat hepatocytes inside artificial cells recover theirviability after implantation into mice?

Factors which can stimulate liver regeneration have been isolated fromthe liver extract and serum of newborn rats or partially hepatectomizedrats. One of these factors has a molecular weight of <10,000 andthe other one, a molecular weight of >100,000 (Michalopouloset al. 1984). Alginate-polylysine-alginate membrane of artificial cellsis only permeable to molecules of up to 64,000molwt (Ito andChang, 1992). Our study (Kashani and Chang, 1988) shows that thehigher molecular weight hepatic growth factor of >100,000m.w. iscontinuously secreted by the hepatocytes inside the artificial cells.Since this type of artificial cells is not permeable to molecules largerthan 64,000, we found that the >100,000m.w. hepatic growth factorcontinues to accumulate inside the artificial cells. Trypan blue stainexclusion only shows the increase in permeability and damage of thehepatocytes and not necessarily death of the hepatocytes. Thus, it islikely that the increasing concentration of this hepatic growth factorinside the artificial cells may have played a role in the regeneration ofthe damaged hepatocytes inside the artificial cells.

Aggregation of artificial cells after implantation

It has been generally accepted that aggregation of artificial cells afterimplantation is due to the problem of biocompatibility. As mentioned


earlier, the aggregation of the artificial cells was due to fibrous coating.We now look at another possible reason. We examined the artificialcells containing rat hepatocytes that have form fibrous aggregatesafter implantation into mice (Wong and Chang, 1991a). In many suchcases, one can find the occasional hepatocytes extruding out of themembrane of the artificial cells after implantation (Fig. 9.5). The mostsevere tissue reaction seems to occur especially at these sites.We then carried out another study on newly prepared alginate-

polylysine-alginate artificial cells containing hepatocytes that have notbeen implanted. Using serial sections, we observed that in a significantnumber of the artificial cells, hepatocytes were extruding out of themembrane or entrapped in themembrane of the artificial cells (Fig. 9.5)(Wong and Chang, 1991a).We hypothesize that this may be one of thecauses of the aggregation of the artificial cells containing hepatocytes.Each artificial cell contains more than 100 hepatocytes. Even whenone hepatocyte out of these is exposed to the surface, the body willgenerate an immune response, resulting in the rejection of the wholeartificial cell. In those cases where the hepatocytes are entrappedin the membrane but not exposed to the outside, weakening of themembrane will result. With time, erosion can result in the hepatocytesbeing exposed to the outside, followed by immunorejection.My analysis of the standard method of preparation shows that with

more than 100 hepatocytes in one artificial cell, the exposure ofone or two hepatocytes to the surface is likely a random process(Fig. 9.5). I therefore devised a two step method to overcome thisproblem.The procedure and principle have been described in detail inChapter 8 and Appendix II (Wong and Chang, 1991b). Thus, extrusionof hepatocytes to the outside and entrapment in the membrane areprevented (Fig. 9.5).

Artificial cells prepared by standard method: cell viabilityafter implantation

Implanted artificial cells were recovered by laparotomy and analyzed(Liu and Chang, 2002). For the artificial cells prepared using thestandard method, from week 2, most of the microcapsules were seenattached to the greater momentum, portal tract, pancreas and surface


Fig. 9.5. Upper: Serial section of artificial cells containing hepatocytesfreshly prepared using the standard method. A hepatocyte is seen to extrudeto the outside and another one is entrapped in the membrane of the artificialcell. Middle: After implantation in mice, fibrous tissue can be seen at sitewhere rat hepatocyte extrudes to the outside. Membrane defects are present14 days after implantation. Lower: Artificial cell containing rat hepatocytesprepared using the new 2-step method show no hepatocytes extrusion orentrapment. No membrane defect was observed 14 days after intraperitonealinjection into mice. (Wong and Chang 1991a, 1991b; Liu and Chang, 2002).


of the liver and the spleen. At week 3, very few intact free artificialcells could be recovered; at week 4, the aggregated microcapsuleswere enclosed by more connective tissue, and some newly formedblood vessels were observed among the aggregates of microcapsules.Under the microscope, macrophages and lymphocytes were foundinfiltrating the capsular membrane (Fig. 9.5). No further morphologicaland viability studies were carried out in these groups after week 4, aslittle or no intact microcapsules could be recovered.

Artificial cells prepared by two-step method: cell viabilityafter implantation

In contrast to the above results, when artificial cells were preparedusing the new two-step method, the following results were obtained(Liu and Chang, 2002). At week 4, more than 90% of transplantedintact microcapsules could be recovered. At 8 weeks, 12 weeks and16 weeks, the recovery rate was 60%, 50%, 20%, respectively. Fromweek 4 on, some artificial cells were found attached to the greateromentum. Microscopic studies showed little or no inflammatorycells infiltration in the recovered microcapsules membrane (Fig.9.5). Viability study shows that after the two-step encapsulation, thehepatocytes viability inside the artificial cells was 74%–78%. In theworst example of artificial cells containing hepatocytes preparedusing the standard method, one could see membrane holes and cellentrapments (Fig. 9.5). Artificial cells prepared using the two stepmethods do not show such imperfections after implantation (Fig. 9.5).The result serve to show that biocompatibility is not the only factorthat causes the aggregation and fibrous enclosure of artificial cellsafter implantation.

9.4. Artificial Cells Containing Hepatocytes orHepatocytes Plus Stem Cells

Introduction

The next step is to try to further increase the duration of function andviability of the hepatocytes inside artificial cells. For this purpose,


we compare artificial cells containing hepatocytes to artificial cellscontaining hepatocytes and stem cells prepared by the two-stepmethod.

Viability of free cells in culture

We first studied plated culture hepatocytes and compare this toplated co-culture of hepatocytes and bone marrow stroma cells (Liuand Chang, 2000). Free hepatocytes are detached 4 h after plating,and reached the optimal growth stage at day 7. At day 14, mosthepatocytes became detached and cell death occurred. In the co-culture of hepatocytes and bone marrow stem cells, the hepatocyteswere maintained for a longer period of time (14–21 days) than those ofthe hepatocyte culture. At day 14, most of the hepatocytes remainedattached, and showed normal growth. This result leads us to the nextstep of coencapsulating hepatocytes and bone marrow stem cells inartificial cells.

In vitro viability of encapsulated cells

We compare the in vitro viability of hepatocytes encapsulated alonein artificial cells with that of hepatocytes co-encapsulated with abone marrow stem cells (Liu and Chang, 2000). They are prepared asdescribed under Methods in Chapter 8. The most striking microscopicobservation is that when only hepatocytes are encapsulated, mostof the cells remain single after encapsulation and during the cultureperiod (Fig. 9.6).When co-encapsulated with bone marrow stem cells,most of the hepatocytes are attached to one another after encapsulationand during the culture period (Fig. 9.6). Mechanical rupturing ofthe artificial cells releases the encapsulated cells for viability studiesusing trypan blue exclusion. After isolation from the intact liver, theviability of the hepatocytes was 85%–90%, and after isolation fromthe femur, the bone marrow stem cells (nucleated cells) was 95%–100%. Immediately after encapsulation, the hepatocytes viability was76%–83%. Starting from day 14 in the culture medium, the viabilityof hepatocytes in co-encapsulation was significantly higher than the


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Most hepatocytes becomedetached with lowerviability

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Hepatocytes in AC

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Fig. 9.6. Comparision between artificial cell containing hepatocytes andartificial cell containing both hepatocytes and bone marrow stem cells. After28 days in culture; Upper : appearance of hepatocytes when encapsulatedalone (left) and in combination with stem cells (right); and Middle : viabilityin culture; Lower : viability after implantation into rats. (Liu and Chang, 2002).


hepatocytes encapsulated alone (P < 0.01) (Fig. 9.6). By the 28th days,the viability of the hepatocytes co-encapsulated with bone marrowcells was maintained at a high level (Fig. 9.6). On the other hand, theviability of the hepatocytes encapsulated alone has fallen to very lowlevels (Fig. 9.6).After implantation, the viability or hepatocytes in 1) artificial cells

contain hepatocytes alone; and 2) artificial cells containing bothhepatocytes and bone marrow stem cells, are as follows. From week 1to week 6 post-transplantation, there was no significant difference inhepatocytes viability between the 2 groups (Fig. 9.6). However, fromweek 7 toweek 16 post-transplantation, the viability of the hepatocytesco-encapsulatedwith bonemarrow stem cells, was significantly higherthan that of the group with only hepatocytes (Fig. 9.6). The average cellnumber in each artificial cell containing both hepatocytes and bonemarrow stem cells remained the same.

Discussion of above results

Artificial cells containing both hepatocytes and bone marrow stemcells make possible a greater hepatocyte viability in culture and afterimplantation. The exact mechanism by which the bone marrow cellsfacilitate the maintenance of the hepatocytes is unknown. When co-encapsulatedwith bonemarrow stemcells, the hepatocyteswere foundto attach to one another, whereas when encapsulated alone, mosthepatocytes remained unattached (Liu and Chang, 2002). The aboveresults seem to show that both cell-cell interactions and secretions fromthebonemarrowstemcellsmaycontribute to this outcome. In addition,other groups have shown that attachment among cells is very importantfor maintaining hepatocytes viability and liver specific function insidethe artificial cells (Theise et al., 2000). Our results show that the co-encapsulation of hepatocytes and bone marrow stem cells improveshepatocytes viability when implanted into normal rats. In addition thenew two-step encapsulation method provides for improved viability ofimplanted hepatocytes.This hepatocyte viabilitywas increased tomorethan 3 months and therefore approached a level such that they couldpossibly be used in our study of liver regeneration. First, a quantitativestudy was carried out to see if this increase in viability corresponds toactual increase in function.


Study using Gunn rat model

We compare the effect of implantation of artificial cells prepared usingthe two-stepmethod and containing 1) no hepatocytes; 2) hepatocytes;and 3) both hepatocytes and bone marrow stem cells (Fig. 9.7) (Liuand Chang, 2003). In the Gunn rats, the blood total bilirubin level was4.4 ± 0.86mg/dl before injection of the artificial cells. In the controlgroups of empty artificial cells, the bilirubin levels increased steadilythroughout the 8 weeks and by week 8, the bilirubin level had reached9mg/dl, about doubled the initial values. For artificial cells containingonly hepatocytes, the plasma bilirubin levels decreased significantly,reaching the lowest level (2.7mg/dl) at week 2 after transplantation.After this, the bilirubin level increased progressively reaching the samelevels as the control groups at week 7 post transplantation. For artificialcells containing both hepatocytes and bone marrow stem cells, thebilirubin levels also decreased to the lowest level at week 2 after

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Fig. 9.7. One intraperitoneal injection into each Gunn rat of (1) controlartificial cells containing no hepatocytes; (2) artificial cells containinghepatocytes; or (3) artificial cells containing both hepatocytes and bonemarrow stem cells. The results on the total systemic bilirubin levels wasfollowed for 70 days. Artificial cells were prepared using the 2-step method.(Liu and Chang, 2003).


transplantation. However, the important difference is that in this case,the level remained significantly lower than that of the control groupup to 8 weeks after injection (Fig. 9.7) (Liu and Chang, 2003).

9.5. Artificial Cells Containing Hepatocytes in Rats with90% of Liver Surgically Removed

Introduction

The above studies show that co-encapsulation of hepatocytes and bonemarrow stem cells inside artificial cells allows us to attain 8 weeks ofboth hepatocyte viability and hepatocyte function. This means that weare ready to proceed to the next step of studying the use of this methodfor liver regeneration. Our original plan was to study the regenerationof liver in rats with 90% of liver surgically removed using the following:1) control with no hepatectomy; 2) control with hepatectomy; 3) freehepatocytes; 4) artificial cells containing hepatocytes; 5) artificial cellscontaining both hepatocytes and bone marrow stem cells; 6) free bonemarrow stem cells; 7) artificial cells containing bone marrow stemcells. We started with (1), (2), (3) and (4) and obtained the followingresults.

Artificial cells prepared using 2-step method on survivalof rats with 90% of liver surgically removed

In the 90% hepatectomy group (PH) and the PH groups that receivedempty control microcapsules, most of the rats died in the firstthree days after hepatectomy. There was no death in the shamcontrol group of rats that did not undergo liver resection. In thetwo groups receiving artificial cells containing hepatocytes or freehepatocytes, the survival rate was not significantly different fromthat of the sham control group (Fig. 9.8) (Liu and Chang, 2006).Free hepatocytes from Wistar rats were not immunorejected by theGunn rats. Thus, both free hepatocytes and artificial cells containinghepatocytes alone enabled the rat liver to regenerate resulting in the


Fig. 9.8. Except for one group with no hepatectomy, the other groups arerats with 90% of their liver removed surgically. One intraperitoneal injectionof (1) free hepatocytes; or (2) hepatocytes in artificial cells (AC) results inincreased in survival rates that are not significantly different from the normalrats. (Liu and Chang, 2005).

recovery of the rats. However, three important points need to beconsidered.

Factors to be considered

(1) Rat liver can regenerate much faster than human liver. The resultsdiscussed earlier in this chapter show that artificial cells prepared usingthe new two step method containing hepatocytes alone can remainviable after implantation for up to 14 days and they can also functioneffectively in maintaining a low bilirubin in the Gunn rats for up to14 days. In humans, liver regeneration takes much longer and 14 daysmay not be sufficient and most likely need up to 8 weeks. Thus, onewould likely need to consider the use of artificial cells containing bothhepatocytes and bone marrow stem cells that have a much longerduration of function and viability after implantation, i.e. up to 8 weeks.


(2) The present rat model with 90% of the liver removed surgicallycan be applied to clinical situations where liver resection is soextensive that it is not compatible with life. This is especially the casein extensive cancer metastasis to the liver or in extensive traumaticinjuries to the liver. However, in the case of fulminant hepatic failure, inaddition to inadequate liver function, there is also the other importantfactor of toxins released from the necrotic liver cells. These toxinscan inhibit the regeneration of the remaining liver tissue, in additionto being toxic to the body. The next chapter describes the use ofan adsorbent artificial cell hemoperfusion device to remove thesetoxins. This results in the removal of the toxins and the recovery ofconsciousness in grade IV hepatic coma patients (Chang, 1972b).However, hemoperfusion only removes the toxins and does not enablethe production of hepatic growth factor to stimulate the regenerationof the liver or render to support the inadequate liver functions. Acombination of hemoperfusion at the beginning to remove the toxinsand use of artificial cells containing hepatocytes and bone marrowstem cell should provide the needed functions.(3) Although artificial cells containing hepatocytes can prevent

immunorejection without the need for immunosuppressant, it isdifficult to obtain hepatocytes from humans. The use of hepatocytesfrom animals can also help prevent immunorejection by using artificialcells. However, since smaller protein molecules like albumin secretedby the enclosed hepatocytes can diffuse out of the artificial cells,there is the potential of immunological problems. On the other hand,obtaining bone marrow from humans for use in other patients is aroutine hematological procedure. Thus, if we can use artificial cellscontaining only bone marrow stem cells, then we would solve manyof the potential problems. Thus, we carried out the next study on thispossibility.

9.6. Artificial Cells Containing Bone Marrow Stem Cells

In this study, we injected artificial cells containing bone marrowstem cells intraperitoneally into 90% hepatectomized rats (Liu and


Fig. 9.9. Survival rates of rats with 90% of liver surgically removed,hepatectomy compared to those without removal of liver tissues (nohepatectomy). One peritoneal injection of artificial cells containing bonemarrow stem cells resulted in the survival of the 90% hepatectomized rats.On the other hand, free bone marrow stem cells did not significantly increasethe survival rates. (Liu and Chang, 2006).

Chang, 2006). We then studied the survival rates of the rats and thetransdifferentiation of bone marrow stem cells.

Viability of isolated cells

About 3 × 108 nucleated BMCs could be obtained from the twofemurs of each rat. BMCs viability after bioencapsulation was 98%.Hepatocytes viability after encapsulation was 82%.

Survival rates of rats with 90% of liver surgically removed

Figure 9.10 shows the survival rates of the following groups: 90%hepatectomized rats receiving 1) no artificial cells as control; 2) freebone marrow stem cells; and 3) artificial cells; that contain bone


LIVER WEIGHTS AFTER 2 WEEKSLiu & Chang; J Liver Transplant (2006)

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Fig. 9.10. This summarizes the liver weights of the rats that have survived2 weeks after 90% of liver in each rat was surgically removed (hepatectomy).In the 3 groups where most of the rats died, those few that survived have amuch lower liver weight. In those groups where all the rats survived after2 weeks, the liver weights are not significantly different from the shamcontrol group. This shows that in these groups, the 10% remaining livershave regenerated to the normal size. The rats in the sham control group didnot have any of their liver removed. (Liu and Chang, 2006.)

marrow stem cells. The 4th group consisted of sham control rats thathave not received hepatectomy. In the 90% hepatectomized controlgroup, and the groups that received free bone marrow stem cells, thesurvival rates were significantly lower than the sham control groupwith most of the mortality occurring in the first three days post-surgery.There was no significant difference in the survival rates between thesham control group and the group receiving artificial cells containingbone marrow stem cells. Thus it would appear that in rats, artificialcells containing bone marrow stem cells are as effective as artificialcells containing hepatocytes in improving the survival rates of 90%hepatectomized rats.


Remnant liver weight

For those rats that survived at week 2 post hepatectomy, the remnantlivers were removed, and the weights were measured. In the 90%hepatectomized rats that received 1) artificial cells containing bonemarrow stem cells; 2) free hepatocytes; or 3) artificial cells containinghepatocytes, the liver wet weights were not significantly different fromthose of the sham control group (Fig. 9.11) (Liu and Chang, 2006).On the other hand, most of the animals did not survive in the 90%hepatectomized rats that 1) received no injection; 2) received controlartificial cells containing no cells; and 3) received free bone marrowstem cells (Fig. 9.11). In those few that survived, the liver weights weremuch lower, but the small number did not allow for statistical analysis.

Blood chemistry

From blood chemistry (Liu and Chang, 2006), it was shown that exceptfor the sham surgery group, the albumin levels in all the other groupsdecreased from day 1 post-surgery. After this, in the group that receivedartificial cells containing hepatocytes, the albumin levels increasedand reached the normal level at day 2 post surgery. In the groupsthat received artificial cells containing bone marrow stem cells, thealbumin only started to increase from day 3 post-surgery and reachedthe pre-surgery level in about one week. Blood levels of ALT andAST increased and reached peak levels at day 1 post-surgery for thecontrol artificial cell group, free bone marrow stem cell group and thecontrol group receiving no treatment. In the groups that received freehepatocytes or artificial cells containing bone marrow stem cells orhepatocytes, the results were different. There were significantly lowerincreases in the ALT, AST levels as compared to the hepatectomizedgroup that received no injection or injection of control artificial cellscontaining no cells.

Plasma hepatic growth factor (HGF) levels

In the hepatectomized groups that received artificial cells containinghepatocytes or artificial cells containing bone marrow stem cells, the


Fig. 9.11. Plasma hepatic growth factor (HGF) followed for those rats thathave survived for 14 days. The results are shown as Mean ± S.D. only forthose groups where all the animals have survived. PH = surgical removal of90% of the liver. BMCs= bone marrow stem cells. H= hepatocytes. Encap=in artificial cells. (Liu and Chang, 2006.)

blood levels of HGF peaked at days 2 and 3 post surgery, and weresignificantly higher than those in the other groups (Liu and Chang,2006). After this, the levels decreased and returned to pre-surgerylevel on day 14. In the free hepatocytes transplantation group, thepeaked level was significantly lower at day 2 post surgery, decreasingto pre-surgery level at day 7. In the control hepatectomized groups thatreceived 1) no treatment 2) artificial cells containing no cells group;or 3) free bone marrow stem cells, the blood HGF levels were muchlower (Fig. 9.11).

Laparotomy and histology

Two weeks after transplantation, laparotomy showed that in the groupthat received artificial cells containing bone marrow stem cells, theartificial cells remained in the peritoneal cavity.They were found freelydistributed throughout the peritoneal cavity or to be behind the liveror under the spleen had adhered or attached to the large omentum.Histology examination of the bone marrow stem cells in the artificial


WBCANTIBODY

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AFP GLYCOGEN

Fig. 9.12. Upper : Immunochemistry stain for hepatocyte markers (CK18and CK8) for albumin production, glycogen production and for AFP. Lower :Possible mechanisms for artificial cells containing bone marrow stem cells inliver regeneration. (Liu and Chang, 2006.)


cells showed that before transplantation most cells were polygonal,star like, some with tail shape cytoplasm (Fig. 9.12). When they wereretrieved two weeks after transplantation, much of cells morphologywas transformed into round or oval shape (Fig. 9.12) (Liu and Chang,2006).

Immunocytochemistry

Hepatocytes recovered from artificial cells show cluster of cellspositively stained with hepatocyte markers CK8 and CK18 and alsoshowing production of albumin ALB (Fig. 9.12), but no cells wasfound to stain positive with AFP. Before implantation, the bonemarrow stem cells recovered from artificial cells did not show positiveimmunochemistry stain. However, when bone marrow stem cellswere recovered from the artificial cells retrieved at week 2 afterimplantation, it was found that scattered cells were positively stainedwith hepatocyte markers CK8 and CK18 and also albumin production(Fig. 9.12). There were also cells that stained positively with AFP(Fig. 9.12 ) (Liu and Chang, 2006).

PAS glycogen stain

PAS staining revealed that hepatocytes recovered from artificial cellsretrieved 2weeks post-transplantationwere positive for PAS, indicatingthese hepatocytes remained capability of glycogen production. In thebone marrow stem cells recovered from artificial cells retrieved twoweeks post-transplantation, there were also cells that stained positivefor PAS, indicating that the cells are capable of producing of glycogen(Fig. 9.12).

Possible mechanisms responsible for recovery of 90%hepatectomized rat model

What are the mechanisms responsible for the recovery of 90%hepatectomized rat model in our study? Most likely two combinedmechanisms are involved 1) The bone marrow stem cells in the


artificial cells release the hepatic growth factors HGFs that stimulatethe regeneration of the remaining 10% liver in the rats that had 90%of the livers surgically removed; and 2) transdifferentiation of the bonemarrow stem cells in the artificial cells into hepatocytes (Fig. 9.12).

(1) Transdifferentiation into hepatocytes. Our immunochemistry stainstudies show that some of the bone marrow stem cells recoveredfrom the artificial cells transdifferentiated into hepatocyte-like cellsthat expressed ALB, CK8, CK 18 and AFP, typical markers ofhepatocytes. They also produced albumin and glycogen. Couldthese transdifferentiated hepatocytes contribute to the recovery ofthe 90%hepatectomized rats?Transdifferentiation into hepatocyte-like cells is not immediate (Lagasse et al., 2000). Thus, the numberof transdifferentiated cells was limited at the beginning and thisalone could not have provided all the liver support observed inour study.

(2) Another possible mechanism involves the hepatic growth factors(HGFs) an important factor in liver regeneration (Rokstad et al.,2002) as well as in stimulation of the transdifferentiation of BMCsinto hepatocytes (Spangrude et al., 1988). The level of HGFincreases in acute or chronic liver failure (Uchida and Weissman,1992). As discussed earlier, there are two subgroups of HGF, onebeing of higher molecular weight of >100,000 and the other ofsmaller molecular weight of<10,000 (Michalopoulos et al., 1984).Alginate-polylysine-alginate membrane artificial cells allow thepassage of molecules of 64,000 or less and retain those >64,000(Ito and Chang, 1992). Our earlier study showed that HGF of>100,000m.w. secreted by hepatocytes were retained and theyaccumulated in the artificial cells, thus helping to increase theregeneration of hepatocytes in the artificial cells (Kashani andChang, 1988). The smaller molecule weight HGF of <10,000 candiffuse out of the artificial cells to stimulate the regeneration ofthe remaining 10% liver mass in the 90% hepatectomized rats.Indeed, Rokstad et al. (2002) showed that certain type of HGF wasable to pass through the APA membrane and these most likely arethose in the <10,000m.w. range. In this regard, our study showed


that in the 90% hepatectomized rats that received artificial cellscontaining bone marrow stem cells, the blood HGF levels weresignificantly higher than those in the other groups, including the90% hepatectomized group (Fig. 9.11). The HGF-like factors weremost likely released from the bone marrow stem cell inside theartificial cells. As a result, the HGF stimulated the remnant liverto regenerate faster in the first days post transplantation beforethere was enough transdifferentiation of stem cells inside theartificial cells into hepatocytes (Fig. 9.12). Further support for thisexplanation is that artificial cells containing bone marrow stemcells stayed in the intraperitoneal cavity throughout the 14 days ofthe study. Thus, the HGF secreted can be drained into the portalcirculation to reach the 10% remaining liver mass to stimulate itsregeneration. On the other hand, intraperitoneal injection of freebone marrow stem cells did not increase the survival rates. This ismost likely because the free bone marrow stem cells are rapidlyremoved from the peritoneal cavity through the lymphatic drainageand anyHGF released does not drain into the portal circulation andstimulate the regeneration of the remnant liver.

9.7. Artificial Cells Containing Stem Cells inRegeneration Medicine

It would appear from the above study that implantation of artificialcells containing bone marrow stem cells results in the regenerationof the 90% hepatectomized liver and the survival of the animal. Thisis as effective as the injection of free hepatocytes or artificial cellscontaining hepatocytes. It is likely that hepatic growth factor HGFplays an important initial role followed by transdifferentiation intohepatocytes. These observations could stimulate further investigationof the potential for an alternative to hepatocytes transplantation for thetreatment of acute liver failure or extensive liver resection. The use ofartificial cells containing stem cells could also be investigated in otherareas of regenerative medicine.

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Artiﬁcial Cells Containing Hepatocytes and/or Stem Cells