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Multiple access diffusion chamber and the cultivation of liver cells
Experimental
Cell Research
MULTIPLE
493
(1966)
ACCESS DIFFUSION CHAMBER AND THE CULTIVATION OF LIVER CELLS
G. S. GERMAIN, Department Detroit,
43, 493-505
HELENE
SCHNEIDER
of Pathology, Wayne State University School Mich., and School of Basic Sciences, University Baptist Memorial Hospital, Memphis,
and E. E. -MUIRHEAD of Medicine, Woman’s Hospital, of Tennessee Medical Units, Tenn., U.S.A.
Received April 5, 1966
ORG.UV culture has been used as an experimental tool in several instances. Although it would be advantageous to have available a reproducible organ culture of human liver, especially for the study of hepatotropic viruses and certain drug injuries, this tissue has been especially difficult to handle in this explanted form [8, 91. At this time we wish to relate experiences with attempts to grow liver tissue and cells in Algire type diffusion chambers [Z] and on the apparent sustenance of human embryonic liver cells protected by such chambers when implanted in the guinea pig. Some observations of dissociated human embryonic liver cells derived from monolayer cultures which formed aggregates typical of the original tissue after varying times in these chambers are presented. This supports the opinion held by others [15] that information indigenous to each cell can direct subsequent histotypic reaggregation and differentiation without the presence of external inducers. More practically, such tissues produced de nouo from cells could serve as a substrate for hepatotropic viruses. The hypothesized approach would be similar to an approach used to demonstrate effects of poliomyelitis virus [5]. A modification of the diffusion chamber to meet the requirements of such a system is also described.
MATERIALS
AND
METHODS
In the initial experiment, a chamber designed by Stone was used. Fragments of liver were obtained from four-day-old Sprague-Dawley rats as well as from rats of varying agesup to adulthood. To insure that the liver tissue was not affected by post mortem changes,it was removed under ether anesthesiabefore the animal was sacrificed. The tissue was minced, washed in Hank’s balanced salt solution [6] and placed in the diffusion chamber which was sealedand implanted in a pocket formed in the 1 Supported by Army Contract Number DA-49-193-Md-2807. Experimental
Cell Research
43
494
G. S. Germain,
H. Schneider
and E. E. Muirhead
abdominal musculature of a host Sprague-Dawley rat by blunt dissection. The time from liver removal to implantation did not exceed 30 min. After a period of 7-21 days, the chambers were removed and the contents sectioned and stained with hematoxylin and eosin. Diffusion chambers contained liver tissue from Sprague-Dawley rats were implanted into guinea pig hosts. The same procedure was followed as with rat hosts with the exception that the diffusion chamber was modified from the original Stone chamber (Fig. la). Chambers were removed 6-18 days after implantation, opened and the contents were sectioned and stained with H & E. Human fetuses were obtained from spontaneous or therapeutic deliveries. The elapsed time from delivery to removal of the liver varied from 2 to 16 hr and was not subject to our control The excised liver was transferred to media 199 without serum where portions were removed and minced for subsequent use in one or all of the following procedures. (1) Minced tissue was suspended in 199 without serum and injected into a chamber until the tissue volume was one-third the capacity of the chamber and was then implanted into the guinea pig. (2) Minced tissue was subjected to trypsinization and agitation in 0.25 per cent trypsin in a Bellco spinner flask for 30 min. The trypsin containing suspended cells was removed and centrifuged at 1500 rpm for 10 min and the supernatant was decanted. The pellet was then resuspended in 5 cc of serum-free 199 and again centrifuged at 1500 rpm for 10 min. This pellet was suspended in 0.2 cc of serum-free 199 and injected in a chamber which was then implanted into a guinea pig. (3) After trypsinization as in the above procedure the pellets derived from the first centrifugation were suspended in 199 +8 per cent or 15 per cent fetal bovine serum and this suspension was used to inoculate glass bottles. After varying periods of cultivation as monolayers the cells were harvested either by scraping into their own media or by trypsinization. In both cases the resulting suspension was centrifuged, the supernate decanted and the pellet resuspended in 1 cc of serum-free 199. Enough of this suspension was drawn into a 1 cc syringe to result in 2.5 -5 x lo8 cells being injected into each chamber. The chambers were then implanted into guinea pigs or in one case into a rat. The implantation site for single chambers in guinea pigs was a small pocket formed between two thin muscle layers in the abdomen. The incision in the overlaying muscle layer was closed with 4-O gut and the skin was closed separately with 4-O ethilon suture. If two connected chambers were to be implanted one chamber was placed subcutaneously on the animal’s side while the additional chamber was placed in the peritoneal cavity through incisions which were subsequently closed with 3-O gut suture. The skin was closed with 4-O ethilon suture. Chambers were removed and placed briefly in saline before the outside membranes were wiped with cotton gauze to remove any adherent host cells. They were then visually inspected for obvious cracks or punctures. If none were present, the whole chamber was placed in buffered formalin for 1 to 2 hr to fix cells in their relative positions. The chambers were then opened and if the original inoculum was minced tissue, it was removed and placed in buffered formalin, but if it was either of the two types of cell suspension, the membranes with attached cells were removed intact from the chamber frame and placed in buffered formalin. All the material from each chamber was serially sectioned and stained with H & E. Experimental
Cell Research
43
Diffusion
Fig. l.-(a) Original modified by drawing chambers connected
chamber
and cultivation
495
of liver cells
stone chamber is a 17 mm x 17 mm x 2 mm Lucite frame. (b) Chamber a length of polyethylene tubing through a hole drilled in the side. (c) Two with polyethylene tubing, one chamber having an entry tube.
Serum-free media was used since it was found that fetal bovine serum elicited strong antibody production even when present in small amounts. It became apparent that in order to inject a virus or drug into the chamber, a technic was needed which did not require the chamber to be moved. Algire [l] and Amos [3] used a hole drilled in the edge of their chamber to facilitate handling. Our modification of this procedure was accomplished by drilling a l/32 inch hole in the chamber side and drawing a length of P.E. 160 l tubing through this hole (Fig. 1 b). The free end of the tube was then sealed and buried in the subcutaneous tissue. To inject into the chamber, a small cutaneous incision was made, the end of the tube exposed, opened and the material was injected through the tube into the chamber. A second variation consisted of two chambers connected by P.E. 160 tubing (Fig. lc), one of which had a second tube for the injection of experimental material and was implanted in the muscle, while the additional chamber was placed in the peritoneal cavity. For all chambers, Millipore HA membranes with a porosity of 0.45 y were used and were attached to the frame with a solution of lucite in acetone. Chambers were pressure-tested for leaks by injecting air through the tube while the chamber was immersed in water. If leakage occurred, it was resealed. Two randomly chosen chambers were pressure tested with water to determine the strength of the frame to membrane seal in order to estimate the rate that media 1 Clay-Adams, 32 - 661807
Inc.,
New
York. Experimental
Cell Research
43
G. S. Germ&,
H. Schneider
and E. E. Muirhead
and tissue could be injected without rupturing the membrane or seal. A syringe was connected to the diffusion chamber and a pressure transducer by a T-tube. The tube, transducer and syringe were carefully filled with water to avoid air bubbles. Pressure was then applied through the syringe and the pressure was read on a polygraph. The chamber withstood a pressure of 0.4 atmospheres as indicated by no subsequent leaks. All chambers were sterilized in ethylene oxide or by soaking in 70 per cent ethyl alcohol for 15 min followed by three rinses in sterile water. RESULTS
All homologous minced transplants of liver into rat hosts were unsuccessful. Fragments removed from the chambers were completely necrotic. These findings were not improved by shortening the duration of implantation to seven days. The intramuscular site for implantation of chambers in rats appeared a poor location as large quantities of exudate and detritus accumulated around the chamber. The success of some heterologous thyroid implants by Stone [ 12, 131 in diffusion chambers into the abdominal muscle of guinea pigs led us to try this host for rat liver. Using this approach, three livers showed some survival of the parenchymal tissue when removed after 7-10 days. In the remaining transplants of 16-18 days, the parenchymal tissue was necrotic, although an outgrowth of tibroblast-like cells in all cases was observed. Survival of minced human embryonic liver tissue in guinea pig hosts was poor after four or five days and non-existant 16 to 24 days post-implantation. A few parenchymal cells and the hematopoietic, islands survived the short duration implants but in those exceeding 12 days no viable parenchymal cells were seen. The hematopoietic islands apparently survived better as they were present in cultures of 15 days but neither parenchymal or hematopoietic cells were present in cultures of 20 to 24 days. Cells placed in chambers immediately after being isolated by trypsinization were the least successful. In the three experiments attempted of 10 to 12 days duration only a very few cells of undetermined type survived. Of the fourteen monolayer cultures transplanted to guinea pigs all survived. Thirteen of these cultures contained red blood cells after this in viva Figs. 2 and 3.-H & E stain. Human embryonic liver in a diffusion chamber. Vascular structures containing ent. x 160.
cells five weeks in culture and three weeks red blood cells and neutrophils are appar-
Fig. 4.-H & E stain. Human embryonic liver cells five weeks in culture and three weeks in a diffusion chamber. A low magnification of the liver-like tissue showing two mitotic figures. x 100. Experimental
Cell
Research
43
Diffusion
chamber
and cultivation
497
of liver cells
Experimental
Cell Research
43
498
G. S. Germain,
H. Schneider
and E. E. Muirhead
cultivation. Two of the latter cultures also had vessel-like structures containing some of the red blood cells. Importantly, three monolayer cultures, isolated from different embryos produced tissue morphologically similar to liver after varying lengths of time in uiuo. These three experiments together with two representative of the remaining eleven are described more fully below. Human embryonic liver cells grown for five weeks in monolayer culture and transferred twice were injected into a chamber. The chamber was then implanted into the abdominal muscle of the guinea pig and left there for three weeks. The material removed from this chamber consisted of cells organized into the following three patterns: blood vessels, liver-like tissue and columnar epithelium (Figs. 2, 3, 4). The columnar epithelium was present as a single aggregate (Fig. 5). The blood vessels contained red blood cells, red blood cell precursors and neutrophils (Figs. 6, 7). The liver-like tissue appeared to have vascular spaces, one of which contained a neutrophi1 (Fig. 8). In one section of this tissue eleven mitotic figures were present, two of which can be seen in Fig. 4. A second monolayer in culture 34 days and subcultured five times produced liver-like tissue whose morphology was more typical than that of the previous culture (Fig. 9). A single capillary-like structure containing red blood cells was also present (Fig. 10). The duration of implantation was ten days. The third monolayer culture was initially grown for three months, then frozen and stored in liquid nitrogen for six months and finally, cultivated for three additional months until in the 9th subculture, it was placed in a diffusion chamber. This culture also produced liver-like tissue after ten days in uiuo (Fig. 11). Some parenchymal cells of this and the former culture contained a yellow pigment. Liver cells in culture for five months resulted, after 23 days in the chamber, in fibroblastic growth only (Fig. 12). This culture also contained blood elements (Fig. 13).
Fig. 5.-H diffusion x 160.
& E stain. Human embryonic liver chamber. A small clump of columnar
Figs. 6 and 7.-H & E stain. blood cell precursors. x 400.
Higher
Fig. 8.-H & E stain. Higher vessel containing a neutrophil.
magnification x 400.
Experimental
Cell Research
43
magnification
cells five weeks in culture and three weeks in a epithelium was also present in this chamber. of vessels
of example
of Fig.
with
a neutrophil
2. Note
and possibly
epithelial-like
red
cells and a
Biffrrsion
chnmber
and culfivation
of liver cells
499
Experimental Cell Reseali92 43
500
G. S. Germain,
Fig. 9.-H & E stain. Human days in a diffusion chamber. Fig. 10.-H & E stain. culture as in Fig. 9. Experimental
embryonic liver The cells formed
A capillary-like
Cell Research
H. Schneider
43
structure
and E. E. Muirhend
cells after 34 days in monolayer a tissue morphologically similar containing
red blood
cells produced
culture to liver.
and 10 x 160.
by the same
Diffusion
chumher
rind
cultivfltion
of liver cells
Fig. Il.-H & E stain. Human embryonic liver cells after about 6 months in monolayer culture followed by 10 days in a diffusion chamber. Cords of cells can be seen which are reminiscent of liver. x 160. Fig. 12.-H a diffusion
& E stain. Human embryonic liver cells after five chamber. Only fibroblastic cells are present. x 40.
months
in culture
Experimental
and 23 days
in
Cell Research 43
G. S. Germnin,
H. Schneider
and E. E. Muirhead
Fig. 13.-H & E. Red blood cells were found in the interstices cells in the same example as in Fig. 12. x 100. Fig.
14.-H
Experimental
& E stain.
A cord
Cell Research
43
of cells with
an associated
present
red blood
cell.
among x 160.
the fibroblastic
Diffusion
chamber
and cultivation
of liver cells
503
In some cases growth in single and double chambers was limited to libroGrowth was improved in double blastic cells adjacent to each membrane. chambers from the normal two layers to three or four cell layers with additional strands traversing the interior of each chamber (Fig. 14). Roughly, this quadrupled the cell population by doubling the membrane area. It is important to note that ten human embryonic liver implants into guinea pigs retained their hematopoietic function while only three of these also had liver-like epithelial outgrowths. Hematopoietic function was assumed if red blood cells were present. This was possible since cultures of other types of non-hematopoietic cells and even some liver cultures did not produce red blood cells after periods in vivo and subsequent processing completely equivalent to the hematopoietic cultures. Mitotic figures were abundant in two cultures after in vivo cultivation and absent in the remaining eleven. The implications of this are not understood. No correlation could be found between the variables, culture age, transfer I.
TABLE Number
of experiment
Origin of donor tissue or cells and initial treatment Host
animal
21
Minced rat liver
Rat
1
10
3
10
14
Trypsinized human embryonic liver
Minced rat liver
Trypsinized human embryonic liver
Minced human embryonic liver
Trypsinized human embryonic liver
Rat
Guinea
Guinea
Guinea
Guinea
pig
pig
pig
pig Days in monolayer culture Age in days of donor rats when liver removed for implantation Days
in chamber
Implantation Number survival
sitea
-
43
4-adult
-
l-20
6-21
21
6-18
10-12
4-24
I.M.
I.P.
I.M.
I.M.
I.M.
0
0
3
0
4
14-180
-
lo-23 I.M.
or I.M.P.
showing
a I.M., Intramuscular implantation plantation. I.M.P., Intramuscular and are connected by a polyethylene tube. 33-
-
661807
in abdominal intraperitoneal
musculature. implantation
14
I.P., Intraperitoneal of two chambers
Experimental
imwhich
Cell Research
43
504
G. S. Germain,
H. Schneider
and E. E. Muirhead
number, embryo age, duration of implantation tissue. The results of these and all other experiments
and production are summarized
of liver-like in Table I.
DISCUSSION
Since liver cells are difficult to grow and maintain in culture the observations herein recorded are encouraging. Cells of an epithelial origin apparently existed in the monolayer cultures as indicated by the organized aggregates having some morphologic features of the tissues of origin when the cultured cells were grown in the diffusion chamber, Others have demonstrated histotypical aggregation of dissociated chick liver cells subsequent to their return to in uivo [15]. In viuo differentiation into capillary-like structures has also been observed [lo]. In a majority of the human liver cultures examined, which were placed in guinea pig hosts and showed survival, erythropoietic elements appeared to continue. That these structures were latent or extant in the original monolayer cultures is likely since observations of this type have been recorded [7, 111. The possibility of latent structures and functions has been stated [4] and these observations argue strongly for such a potential. It must be emphasized that these monolayer cultures had not entered a stage of rapid proliferation and hence were probably diploid although this has not yet been determined. The attachment of a polyethylene tube ot the chamber made possible several approaches. The chamber contents could be sampled with needles or loops without disturbing its position. More accurate pressure testing of the chamber for leaks was possible. A series of chambers could be connected together, permitting the introduction of different material in each chamber to observe possible mutual relationships. The tubing could be used as a handle, thus preventing damage to membranes by fingers or forceps. Most important, the cells could be injected directly while the chamber was in situ within tissues. Two chambers connected by a tube, one placed in muscle and the other in the peritoneal cavity, had more luxuriant growth. This may have been due to the greater surface area. It could also have been due to a greater flow of fluids, since the peritoneal fluid is low in proteins [ 141 while the serum about the muscle-imbedded chamber is high in proteins. Such a flow could improve the removal of metabolic wastes and improve nutrition.
Experimental
Cell Research
43
Diffussion
chamber
and cultivation
of liver cells
505
SUMMARY
Attempts were made to grow rat and human liver tissue or ceils in diffusion chambers for use as a system to study viral agents and drug injury. Rat tissue in rat or guinea pig hosts was generally unsuccessful. Human embryonic liver cells pre-grown in monolayer culture survived well while in the diffusion chamber within guinea pig hosts, forming cell aggregates that had morphology reminiscent of the original tissue. Erythropoiesis was displayed. The addition of a polyethylene tube to facilitate introduction of agents resulted in a more versatile chamber. Two chambers connected by polyethylene tubing and implanted in different regions, improved the appearance of the contained cells. REFERENCES 1. ALGIRE, G. H., BORDEN, M. L. and EVANS, V. J., J. Natl Cancer Inst. 20, 1187 (1958). ALGIRE, G. H., WEAVER, J. M. and PREHN, Il. L.. ibid. 15. 493 11954). AMOS, D. B. and WAKEFIELD, J. D., .I. N&l Can& Inst. il, 655 (1958). DAVIDSON, E. H., in Advances in Genetics, Vol. 12, p. 189. Academic Press, New York, ESCHENBRENNER, A. B. and FRANCIS, R. D., (Abstract) Fed. Proc. 15, 514 (1956). HANKS, J. H., in W. F. SCHERER (ed.), An Introduction to Cell and Tissue Culture, Burgess Publications, Minneapolis, 1955. 7. HAYFLICK, L. and MOORHEAD, P. S., Expff Cell Res. 25, 585 (1961). 8. HILLIS, W. D. and BANG, F. B., Expfl Cell Res. 26, 9 (1962). 9. INGRAM, R. L., Expff Cell Res. 28, 3’70 (1962). 10. PETRAKIS, N. L., DAVIS, M. and LUCIA, S. P., Blood 17, 109 (1961). 11. SCHNEIDER, H., MUIRHEAD, E. E. and ZYDECK, F. A., Exptl Cell Res. 30,449 (1963). 12. STONE, H. B. and KENNEDY, W. J., Ann. Surg. 155,623 (1962).
2. 3. 4. 5. 6.
13. --
14. 15.
ibid.
159, 645 (1964).
STRAUBE, R. L., Cancer WEISS, P. and TAYLOR,
Res. 18, 57 (1958). C., Proc. Nat2 Acad.
A.
1964. p. 5.
Sci. 46, 1177 (1960).
Experimental
Cell
Research
43
Cell Research
MULTIPLE
493
(1966)
ACCESS DIFFUSION CHAMBER AND THE CULTIVATION OF LIVER CELLS
G. S. GERMAIN, Department Detroit,
43, 493-505
HELENE
SCHNEIDER
of Pathology, Wayne State University School Mich., and School of Basic Sciences, University Baptist Memorial Hospital, Memphis,
and E. E. -MUIRHEAD of Medicine, Woman’s Hospital, of Tennessee Medical Units, Tenn., U.S.A.
Received April 5, 1966
ORG.UV culture has been used as an experimental tool in several instances. Although it would be advantageous to have available a reproducible organ culture of human liver, especially for the study of hepatotropic viruses and certain drug injuries, this tissue has been especially difficult to handle in this explanted form [8, 91. At this time we wish to relate experiences with attempts to grow liver tissue and cells in Algire type diffusion chambers [Z] and on the apparent sustenance of human embryonic liver cells protected by such chambers when implanted in the guinea pig. Some observations of dissociated human embryonic liver cells derived from monolayer cultures which formed aggregates typical of the original tissue after varying times in these chambers are presented. This supports the opinion held by others [15] that information indigenous to each cell can direct subsequent histotypic reaggregation and differentiation without the presence of external inducers. More practically, such tissues produced de nouo from cells could serve as a substrate for hepatotropic viruses. The hypothesized approach would be similar to an approach used to demonstrate effects of poliomyelitis virus [5]. A modification of the diffusion chamber to meet the requirements of such a system is also described.
MATERIALS
AND
METHODS
In the initial experiment, a chamber designed by Stone was used. Fragments of liver were obtained from four-day-old Sprague-Dawley rats as well as from rats of varying agesup to adulthood. To insure that the liver tissue was not affected by post mortem changes,it was removed under ether anesthesiabefore the animal was sacrificed. The tissue was minced, washed in Hank’s balanced salt solution [6] and placed in the diffusion chamber which was sealedand implanted in a pocket formed in the 1 Supported by Army Contract Number DA-49-193-Md-2807. Experimental
Cell Research
43
494
G. S. Germain,
H. Schneider
and E. E. Muirhead
abdominal musculature of a host Sprague-Dawley rat by blunt dissection. The time from liver removal to implantation did not exceed 30 min. After a period of 7-21 days, the chambers were removed and the contents sectioned and stained with hematoxylin and eosin. Diffusion chambers contained liver tissue from Sprague-Dawley rats were implanted into guinea pig hosts. The same procedure was followed as with rat hosts with the exception that the diffusion chamber was modified from the original Stone chamber (Fig. la). Chambers were removed 6-18 days after implantation, opened and the contents were sectioned and stained with H & E. Human fetuses were obtained from spontaneous or therapeutic deliveries. The elapsed time from delivery to removal of the liver varied from 2 to 16 hr and was not subject to our control The excised liver was transferred to media 199 without serum where portions were removed and minced for subsequent use in one or all of the following procedures. (1) Minced tissue was suspended in 199 without serum and injected into a chamber until the tissue volume was one-third the capacity of the chamber and was then implanted into the guinea pig. (2) Minced tissue was subjected to trypsinization and agitation in 0.25 per cent trypsin in a Bellco spinner flask for 30 min. The trypsin containing suspended cells was removed and centrifuged at 1500 rpm for 10 min and the supernatant was decanted. The pellet was then resuspended in 5 cc of serum-free 199 and again centrifuged at 1500 rpm for 10 min. This pellet was suspended in 0.2 cc of serum-free 199 and injected in a chamber which was then implanted into a guinea pig. (3) After trypsinization as in the above procedure the pellets derived from the first centrifugation were suspended in 199 +8 per cent or 15 per cent fetal bovine serum and this suspension was used to inoculate glass bottles. After varying periods of cultivation as monolayers the cells were harvested either by scraping into their own media or by trypsinization. In both cases the resulting suspension was centrifuged, the supernate decanted and the pellet resuspended in 1 cc of serum-free 199. Enough of this suspension was drawn into a 1 cc syringe to result in 2.5 -5 x lo8 cells being injected into each chamber. The chambers were then implanted into guinea pigs or in one case into a rat. The implantation site for single chambers in guinea pigs was a small pocket formed between two thin muscle layers in the abdomen. The incision in the overlaying muscle layer was closed with 4-O gut and the skin was closed separately with 4-O ethilon suture. If two connected chambers were to be implanted one chamber was placed subcutaneously on the animal’s side while the additional chamber was placed in the peritoneal cavity through incisions which were subsequently closed with 3-O gut suture. The skin was closed with 4-O ethilon suture. Chambers were removed and placed briefly in saline before the outside membranes were wiped with cotton gauze to remove any adherent host cells. They were then visually inspected for obvious cracks or punctures. If none were present, the whole chamber was placed in buffered formalin for 1 to 2 hr to fix cells in their relative positions. The chambers were then opened and if the original inoculum was minced tissue, it was removed and placed in buffered formalin, but if it was either of the two types of cell suspension, the membranes with attached cells were removed intact from the chamber frame and placed in buffered formalin. All the material from each chamber was serially sectioned and stained with H & E. Experimental
Cell Research
43
Diffusion
Fig. l.-(a) Original modified by drawing chambers connected
chamber
and cultivation
495
of liver cells
stone chamber is a 17 mm x 17 mm x 2 mm Lucite frame. (b) Chamber a length of polyethylene tubing through a hole drilled in the side. (c) Two with polyethylene tubing, one chamber having an entry tube.
Serum-free media was used since it was found that fetal bovine serum elicited strong antibody production even when present in small amounts. It became apparent that in order to inject a virus or drug into the chamber, a technic was needed which did not require the chamber to be moved. Algire [l] and Amos [3] used a hole drilled in the edge of their chamber to facilitate handling. Our modification of this procedure was accomplished by drilling a l/32 inch hole in the chamber side and drawing a length of P.E. 160 l tubing through this hole (Fig. 1 b). The free end of the tube was then sealed and buried in the subcutaneous tissue. To inject into the chamber, a small cutaneous incision was made, the end of the tube exposed, opened and the material was injected through the tube into the chamber. A second variation consisted of two chambers connected by P.E. 160 tubing (Fig. lc), one of which had a second tube for the injection of experimental material and was implanted in the muscle, while the additional chamber was placed in the peritoneal cavity. For all chambers, Millipore HA membranes with a porosity of 0.45 y were used and were attached to the frame with a solution of lucite in acetone. Chambers were pressure-tested for leaks by injecting air through the tube while the chamber was immersed in water. If leakage occurred, it was resealed. Two randomly chosen chambers were pressure tested with water to determine the strength of the frame to membrane seal in order to estimate the rate that media 1 Clay-Adams, 32 - 661807
Inc.,
New
York. Experimental
Cell Research
43
G. S. Germ&,
H. Schneider
and E. E. Muirhead
and tissue could be injected without rupturing the membrane or seal. A syringe was connected to the diffusion chamber and a pressure transducer by a T-tube. The tube, transducer and syringe were carefully filled with water to avoid air bubbles. Pressure was then applied through the syringe and the pressure was read on a polygraph. The chamber withstood a pressure of 0.4 atmospheres as indicated by no subsequent leaks. All chambers were sterilized in ethylene oxide or by soaking in 70 per cent ethyl alcohol for 15 min followed by three rinses in sterile water. RESULTS
All homologous minced transplants of liver into rat hosts were unsuccessful. Fragments removed from the chambers were completely necrotic. These findings were not improved by shortening the duration of implantation to seven days. The intramuscular site for implantation of chambers in rats appeared a poor location as large quantities of exudate and detritus accumulated around the chamber. The success of some heterologous thyroid implants by Stone [ 12, 131 in diffusion chambers into the abdominal muscle of guinea pigs led us to try this host for rat liver. Using this approach, three livers showed some survival of the parenchymal tissue when removed after 7-10 days. In the remaining transplants of 16-18 days, the parenchymal tissue was necrotic, although an outgrowth of tibroblast-like cells in all cases was observed. Survival of minced human embryonic liver tissue in guinea pig hosts was poor after four or five days and non-existant 16 to 24 days post-implantation. A few parenchymal cells and the hematopoietic, islands survived the short duration implants but in those exceeding 12 days no viable parenchymal cells were seen. The hematopoietic islands apparently survived better as they were present in cultures of 15 days but neither parenchymal or hematopoietic cells were present in cultures of 20 to 24 days. Cells placed in chambers immediately after being isolated by trypsinization were the least successful. In the three experiments attempted of 10 to 12 days duration only a very few cells of undetermined type survived. Of the fourteen monolayer cultures transplanted to guinea pigs all survived. Thirteen of these cultures contained red blood cells after this in viva Figs. 2 and 3.-H & E stain. Human embryonic liver in a diffusion chamber. Vascular structures containing ent. x 160.
cells five weeks in culture and three weeks red blood cells and neutrophils are appar-
Fig. 4.-H & E stain. Human embryonic liver cells five weeks in culture and three weeks in a diffusion chamber. A low magnification of the liver-like tissue showing two mitotic figures. x 100. Experimental
Cell
Research
43
Diffusion
chamber
and cultivation
497
of liver cells
Experimental
Cell Research
43
498
G. S. Germain,
H. Schneider
and E. E. Muirhead
cultivation. Two of the latter cultures also had vessel-like structures containing some of the red blood cells. Importantly, three monolayer cultures, isolated from different embryos produced tissue morphologically similar to liver after varying lengths of time in uiuo. These three experiments together with two representative of the remaining eleven are described more fully below. Human embryonic liver cells grown for five weeks in monolayer culture and transferred twice were injected into a chamber. The chamber was then implanted into the abdominal muscle of the guinea pig and left there for three weeks. The material removed from this chamber consisted of cells organized into the following three patterns: blood vessels, liver-like tissue and columnar epithelium (Figs. 2, 3, 4). The columnar epithelium was present as a single aggregate (Fig. 5). The blood vessels contained red blood cells, red blood cell precursors and neutrophils (Figs. 6, 7). The liver-like tissue appeared to have vascular spaces, one of which contained a neutrophi1 (Fig. 8). In one section of this tissue eleven mitotic figures were present, two of which can be seen in Fig. 4. A second monolayer in culture 34 days and subcultured five times produced liver-like tissue whose morphology was more typical than that of the previous culture (Fig. 9). A single capillary-like structure containing red blood cells was also present (Fig. 10). The duration of implantation was ten days. The third monolayer culture was initially grown for three months, then frozen and stored in liquid nitrogen for six months and finally, cultivated for three additional months until in the 9th subculture, it was placed in a diffusion chamber. This culture also produced liver-like tissue after ten days in uiuo (Fig. 11). Some parenchymal cells of this and the former culture contained a yellow pigment. Liver cells in culture for five months resulted, after 23 days in the chamber, in fibroblastic growth only (Fig. 12). This culture also contained blood elements (Fig. 13).
Fig. 5.-H diffusion x 160.
& E stain. Human embryonic liver chamber. A small clump of columnar
Figs. 6 and 7.-H & E stain. blood cell precursors. x 400.
Higher
Fig. 8.-H & E stain. Higher vessel containing a neutrophil.
magnification x 400.
Experimental
Cell Research
43
magnification
cells five weeks in culture and three weeks in a epithelium was also present in this chamber. of vessels
of example
of Fig.
with
a neutrophil
2. Note
and possibly
epithelial-like
red
cells and a
Biffrrsion
chnmber
and culfivation
of liver cells
499
Experimental Cell Reseali92 43
500
G. S. Germain,
Fig. 9.-H & E stain. Human days in a diffusion chamber. Fig. 10.-H & E stain. culture as in Fig. 9. Experimental
embryonic liver The cells formed
A capillary-like
Cell Research
H. Schneider
43
structure
and E. E. Muirhend
cells after 34 days in monolayer a tissue morphologically similar containing
red blood
cells produced
culture to liver.
and 10 x 160.
by the same
Diffusion
chumher
rind
cultivfltion
of liver cells
Fig. Il.-H & E stain. Human embryonic liver cells after about 6 months in monolayer culture followed by 10 days in a diffusion chamber. Cords of cells can be seen which are reminiscent of liver. x 160. Fig. 12.-H a diffusion
& E stain. Human embryonic liver cells after five chamber. Only fibroblastic cells are present. x 40.
months
in culture
Experimental
and 23 days
in
Cell Research 43
G. S. Germnin,
H. Schneider
and E. E. Muirhead
Fig. 13.-H & E. Red blood cells were found in the interstices cells in the same example as in Fig. 12. x 100. Fig.
14.-H
Experimental
& E stain.
A cord
Cell Research
43
of cells with
an associated
present
red blood
cell.
among x 160.
the fibroblastic
Diffusion
chamber
and cultivation
of liver cells
503
In some cases growth in single and double chambers was limited to libroGrowth was improved in double blastic cells adjacent to each membrane. chambers from the normal two layers to three or four cell layers with additional strands traversing the interior of each chamber (Fig. 14). Roughly, this quadrupled the cell population by doubling the membrane area. It is important to note that ten human embryonic liver implants into guinea pigs retained their hematopoietic function while only three of these also had liver-like epithelial outgrowths. Hematopoietic function was assumed if red blood cells were present. This was possible since cultures of other types of non-hematopoietic cells and even some liver cultures did not produce red blood cells after periods in vivo and subsequent processing completely equivalent to the hematopoietic cultures. Mitotic figures were abundant in two cultures after in vivo cultivation and absent in the remaining eleven. The implications of this are not understood. No correlation could be found between the variables, culture age, transfer I.
TABLE Number
of experiment
Origin of donor tissue or cells and initial treatment Host
animal
21
Minced rat liver
Rat
1
10
3
10
14
Trypsinized human embryonic liver
Minced rat liver
Trypsinized human embryonic liver
Minced human embryonic liver
Trypsinized human embryonic liver
Rat
Guinea
Guinea
Guinea
Guinea
pig
pig
pig
pig Days in monolayer culture Age in days of donor rats when liver removed for implantation Days
in chamber
Implantation Number survival
sitea
-
43
4-adult
-
l-20
6-21
21
6-18
10-12
4-24
I.M.
I.P.
I.M.
I.M.
I.M.
0
0
3
0
4
14-180
-
lo-23 I.M.
or I.M.P.
showing
a I.M., Intramuscular implantation plantation. I.M.P., Intramuscular and are connected by a polyethylene tube. 33-
-
661807
in abdominal intraperitoneal
musculature. implantation
14
I.P., Intraperitoneal of two chambers
Experimental
imwhich
Cell Research
43
504
G. S. Germain,
H. Schneider
and E. E. Muirhead
number, embryo age, duration of implantation tissue. The results of these and all other experiments
and production are summarized
of liver-like in Table I.
DISCUSSION
Since liver cells are difficult to grow and maintain in culture the observations herein recorded are encouraging. Cells of an epithelial origin apparently existed in the monolayer cultures as indicated by the organized aggregates having some morphologic features of the tissues of origin when the cultured cells were grown in the diffusion chamber, Others have demonstrated histotypical aggregation of dissociated chick liver cells subsequent to their return to in uivo [15]. In viuo differentiation into capillary-like structures has also been observed [lo]. In a majority of the human liver cultures examined, which were placed in guinea pig hosts and showed survival, erythropoietic elements appeared to continue. That these structures were latent or extant in the original monolayer cultures is likely since observations of this type have been recorded [7, 111. The possibility of latent structures and functions has been stated [4] and these observations argue strongly for such a potential. It must be emphasized that these monolayer cultures had not entered a stage of rapid proliferation and hence were probably diploid although this has not yet been determined. The attachment of a polyethylene tube ot the chamber made possible several approaches. The chamber contents could be sampled with needles or loops without disturbing its position. More accurate pressure testing of the chamber for leaks was possible. A series of chambers could be connected together, permitting the introduction of different material in each chamber to observe possible mutual relationships. The tubing could be used as a handle, thus preventing damage to membranes by fingers or forceps. Most important, the cells could be injected directly while the chamber was in situ within tissues. Two chambers connected by a tube, one placed in muscle and the other in the peritoneal cavity, had more luxuriant growth. This may have been due to the greater surface area. It could also have been due to a greater flow of fluids, since the peritoneal fluid is low in proteins [ 141 while the serum about the muscle-imbedded chamber is high in proteins. Such a flow could improve the removal of metabolic wastes and improve nutrition.
Experimental
Cell Research
43
Diffussion
chamber
and cultivation
of liver cells
505
SUMMARY
Attempts were made to grow rat and human liver tissue or ceils in diffusion chambers for use as a system to study viral agents and drug injury. Rat tissue in rat or guinea pig hosts was generally unsuccessful. Human embryonic liver cells pre-grown in monolayer culture survived well while in the diffusion chamber within guinea pig hosts, forming cell aggregates that had morphology reminiscent of the original tissue. Erythropoiesis was displayed. The addition of a polyethylene tube to facilitate introduction of agents resulted in a more versatile chamber. Two chambers connected by polyethylene tubing and implanted in different regions, improved the appearance of the contained cells. REFERENCES 1. ALGIRE, G. H., BORDEN, M. L. and EVANS, V. J., J. Natl Cancer Inst. 20, 1187 (1958). ALGIRE, G. H., WEAVER, J. M. and PREHN, Il. L.. ibid. 15. 493 11954). AMOS, D. B. and WAKEFIELD, J. D., .I. N&l Can& Inst. il, 655 (1958). DAVIDSON, E. H., in Advances in Genetics, Vol. 12, p. 189. Academic Press, New York, ESCHENBRENNER, A. B. and FRANCIS, R. D., (Abstract) Fed. Proc. 15, 514 (1956). HANKS, J. H., in W. F. SCHERER (ed.), An Introduction to Cell and Tissue Culture, Burgess Publications, Minneapolis, 1955. 7. HAYFLICK, L. and MOORHEAD, P. S., Expff Cell Res. 25, 585 (1961). 8. HILLIS, W. D. and BANG, F. B., Expfl Cell Res. 26, 9 (1962). 9. INGRAM, R. L., Expff Cell Res. 28, 3’70 (1962). 10. PETRAKIS, N. L., DAVIS, M. and LUCIA, S. P., Blood 17, 109 (1961). 11. SCHNEIDER, H., MUIRHEAD, E. E. and ZYDECK, F. A., Exptl Cell Res. 30,449 (1963). 12. STONE, H. B. and KENNEDY, W. J., Ann. Surg. 155,623 (1962).
2. 3. 4. 5. 6.
13. --
14. 15.
ibid.
159, 645 (1964).
STRAUBE, R. L., Cancer WEISS, P. and TAYLOR,
Res. 18, 57 (1958). C., Proc. Nat2 Acad.
A.
1964. p. 5.
Sci. 46, 1177 (1960).
Experimental
Cell
Research
43