preparation of single-cell suspensions from normal liver ... · hanks' solution (30)...

[CANCER RESEARCH 32, 2568-2576,November 1972]

Preparation of Single-Cell Suspensions from Normal Liver,Regenerating Liver, and Morris Hepatomas 9121 and 5123tc'

Matthias MÃ¼ller,Margot Schreiber, JÃ¼rgenKartenbeck, and Gerhard Schreiber

Biochemisches Institut Â¡M.M., M. S., G. S./ und Institut Biologie II, Abteilung fÃ¼rZellbiologie fJ. K./, UniversitÃ¤tfreiburg im Breisgau, Freiburgim Breisgau, Germany

SUMMARY

Suspensions of single cells were prepared from normal adultrat liver either by mincing the tissue and incubating it invarious concentrations of sodium tetraphenylborate, ethylene-diaminetetraacetate, and 0.1% hyaluronidase plus 0.05%collagenase or by continuous recirculating perfusion of theliver with 0.1% hyaluronidase plus 0.05% collagenase. Only thelatter method produced cells in good yield that were notstained with trypan blue. Incorporation of L-leucine-l-'4C

into protein was up to 60 times higher in cells prepared bycontinuous perfusion with hyaluronidase plus collagenase thanin cells prepared by incubation of the minced tissue in sodiumtetraphenylborate or in hyaluronidase plus collagenase. Livercells prepared by continuous perfusion had a better preservedultrastructure than cells obtained by the other methods.

In regenerating liver, cells that were not stained with trypanblue were obtained by the two methods with hyaluronidaseplus collagenase, the yield being highest after continuousperfusion.

In Morris hepatomas 9121 and 5123tc, continuous perfusion could not be established. However, mincing thetissue and incubating it in hyaluronidase plus collagenaseyielded cells with an intact ultrastructure. Those cells excludedtrypan blue. The amount of the incorporation of L-leucine-1-14C into protein was about 300 times higher than in cells

prepared by sodium tetraphenylborate.

INTRODUCTION

Mammalian cell cultures provide well-defined conditions formetabolic studies. However, it has not yet been possible toobtain cell cultures of normal adult parenchymal liver cells,whereas preparation of suspensions of single liver cells hasbeen described repeatedly (e.g., Ref. 46).

In single cells from rat and mouse liver prepared bymechanical disruption with and without addition ofcalcium-chelating agents or acid salt solutions (1, 8, 15, 39,46,53, 54, 62, 68, 85), lack of aerobic glycolysis (3, 23, 57) andof endogenous respiration (52, 57, 76, 83); impairment of thesynthesis of protein (25), cholesterol (56). and RNA (47);failure of enzyme induction by cortisol (85); leakage of

1This work was supported by Grants Sehr 116/5 and Fr 308/6 fromthe Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany, andthe Stiftung Volkswagenwerk, Hannover, Germany.

Received August 25, 1971 ; accepted August 11, 1972.

enzymes (3,16,19, 23,34, 50, 81, 82,85,92), potassium ions(23, 29, 51, 67), and NAD and ADP (28, 43); an increasedpermeability to calcium ions (3, 23) and to substances of low(48, 78) and high (77,79) molecular weight; and staining withtrypan blue (21, 60) were observed. Furthermore, duringpreparation, destruction of the plasma membrane and of othercell organelles was demonstrated (2, 7, 10, 13, 15, 26, 66, 76).Only in calcium-free media or in media with cofactors added,could respiration (3, 23, 33, 39, 43, 45, 51, 68, 69, 83, 84),synthesis of lipids and cholesterol (10, 41, 42, 60), high ratesof protein (11, 60, 76) and RNA synthesis (76), and oxidationof glucose and fatty acids (21, 22) be observed. Dispersion ofthe cells in polyvinyl alcohol inhibited leakage of enzymes(19) but diminished the rate of respiration (81).

Other preparation methods were based on the proteolyticcleavage of the intercellular matrix. With trypsin, alteration ofthe cell surface (2, 55, 58), deformability of cells (90),increase of permeability (29), and adsorption of trypsin to thecell surface (70) were reported. However, with the use ofhyaluronidase and collagenase, described previously (91), livercells with intact ultrastructure were isolated from rats (38) andmice (20). They synthesized protein (9, 49), RNA (49), andlipids (18, 49), and respired (40, 49) at higher rates thanmechanically prepared cells. Calcium ions stimulated therespiration (40) and the synthesis of protein and lipids (60).Dexamethasone induced tyrosine aminotransferase (32). Thedisadvantage of the new preparation procedure was the lowyield, but continuous recirculating perfusion of the liverimproved the recovery considerably (6, 44). Leakage ofenzymes was lower than in cells prepared with other methods(19). Furthermore, glycolysis (6), gluconeogenesis (6, 80), andalbumin synthesis (89) were demonstrated. Chylomicrons(24), ethanol metabolism (4, 5), and regulation of lipidsynthesis (12) were studied. The only observed damage was aloss of potassium ions (6).

Besides hyaluronidase and collagenase, other dissociatingagents are still in use. EDTA plus lysozyme was reported to bemore effective than other dissociating agents, e.g., hyaluronidase plus collagenase (36, 37). The previously describeddispersion of rat liver cells in TPB2 (71-73) was claimed to be

the only method producing cells that could be grown inculture (14,27).

In this paper, the main methods used recently forpreparation of isolated liver cells are compared on the basis of

2The abbreviation used is: TPB, sodium tetraphenylborate.

2568 CANCER RESEARCH VOL. 32

on April 30, 2020. © 1972 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

Single-Cell Suspensions from Liver and Hepatomas

trypan blue exclusion, of incorporation of L-leucine-l-l4C

into protein, and of preservation of the ultrastructure.Hyaluronidase plus collagenase were found to be superior toother dissociating agents. The procedure could also be appliedsuccessfully to regenerating rat liver and to transplantablehepatomas.

MATERIALS AND METHODS

Chemicals and Isotopes. Hyaluronidase (type I) andneuraminidase (types V and VI) were purchased from SigmaChemical Company (St. Louis, Mo.), and collagenase was fromSigma or from Th. Schuchardt, Chemische Fabrik (MÃ¼nchen,Germany) (Table 1, No. 10, Morris hepatoma 5123tc, firstexperiment) or from Serva, Feinbiochemica GmbH and Co.(Heidelberg, Germany) (Table 1, No. 10, Morris hepatoma9121, both experiments). TPB, analytical grade, was purchasedfrom Sigma or Fluka AG, Chemische Fabrik (Buchs,Switzerland), and lysozyme was from Merck (Darmstadt,Germany). Bovine serum albumin, highest grade, was obtainedfrom Behringwerke AG (Marburg/Lahn, Germany).L-Leucine-l-14C, 62 mCi/mmole, was bought from the

Radiochemical Centre (Amersham, England).Animals, Tumors, and Partial Hepatectomy. ACI and

Buffalo rats of both sexes were used, the body weights rangingfrom 180 to 300 g. Animals were kept as previously described(75). Source and retransplantation of tumors are publishedelsewhere (74). Morris 9121 hepatomas were in generations41, 42, and 47 and 5123tc hepatomas were in generations 98,102, and 110. Hepatoma cell suspensions were prepared fromtumors 3 to 4 weeks after transplantation.

Partial hepatectomies were performed according to themethod of Higgins and Anderson (35). Suspensions ofregenerating liver cells were prepared 48 hr after partialhepatectomy.

Media for Dissociation and Incubation. The TPB dissociation medium was the same as that of Gerschenson andCasanello (27). Hyaluronidase, collagenase, neuraminidase, andEDTA with or without lysozyme were dissolved in a modifiedHanks' solution (30) consisting of 138.9 mM NaCl, 5.37 mM

KC1, 0.81 mM MgSC-4, 0.337 mM Na2HP04, 0.441 mMKH2P04, and 5.5 mM glucose. To each 100 ml, 2.5 ml of a153.6 mM NaHC03 solution were added.

For staining, cells were suspended in a 5.2 mM solution oftrypan blue in modified Waymouth medium (88) consisting of103 mM NaCl, 2 mM KC1, 0.82 mM CaCl2, 1.18 mM MgCl2,0.81 mM MgSO4, 2.11 mM Na2HP04, 0.59 mM KH2P04,26.7 mM NaHC03, 27.8 mM glucose, 0.1 mM ascorbic acid,0.05 mM glutathione, 1.8 mM choline-HCl, 0.18 mMhypoxanthine, 0.03 mM thiamine-HCl, 0.002 mM calcium

pantothenate, 0.003 mM riboflavin, 0.003 mMpyridoxine-HCl, 0.0008 mM folie acid, 0.00008 mM biotin,0.005 mM myo-inositol, 0.008 mM nicotinamide, 0.00015 mMvitamin BÃ•2,0.57 mM cysteine-HCl, 2.38 mM glutamine, 1.42mM L-lysine-HCl, 0.8 mM L-histidine-HCl, 1.02 mML-glutamic acid, 0.64 mM L-threonine, 0.36 mML-arginine -HCl, 0.55 mM L-valine, 0.46 mM L-aspartic acid,0.66 mM glycine, 0.44 mM L-proline, 0.34 mM L-methionine,

0.3 mM L-phenylalanine, 0.22 mM L-tyrosine, 0.2 mML-tryptophan, 0.19 mM L-isoleucine, 0.006 mM L-cystine,0.56 mM alanine, 0.21 mM serine, 10% calf serum, 0.00288%sodium penicillin G, 0.00012% potassium penicillin G, and0.01% streptomycin. For studies on protein synthesis and onultrastructure, cells were suspended in nonmodified Hanks'

solution (30) containing the amino acids according to thelisting of Waymouth (88), 0.02% heparin, 2.5% bovine serumalbumin, and antibiotics as described above. The pH wasadjusted to 7.4 unless stated otherwise.

Preparation of Liver Cell Suspensions by Shaking. After theabdomen and chest of the rat were opened under etheranesthesia, the liver was perfused for about 1 min via thethoracic portion of the inferior vena cava with 20 ml ofdissociation medium at 37Â°.The liver was excised, weighed,

and minced with scissors in 10 ml of ice-cold dissociationmedium into pieces of about 10 cu mm. After being drainedon a plastic tea sieve, the pieces of tissue were resuspended in50 ml of cold, fresh dissociation medium and placed on iceuntil used. Suspensions were incubated at 37Â° under an

atmosphere of 95% 02 and 5% C02 with continuous shaking.After 20 min, nondissociated pieces of tissue were separatedby passing the suspension through a tea sieve and incubated in50 ml of fresh dissociation medium. This was repeated 3 times.Cells were collected from each filtrate by centrifuging for 10min at 30 to 70 X g. For washing, the pellet was resuspendedin 10 ml of incubation medium, centrifuged again, andresuspended in 50 ml of incubation medium to give the finalcell suspensions.

Preparation of Liver Cell Suspensions by ContinuousRecirculating Perfusion. The technique of Berry and Friend(6) with the modifications described previously (89) was used.Cells were washed once with incubation medium.

Preparation of Tumor Cells. Under ether anesthesia, the skinof both hind legs and of the lower abdomen was removed andthe abdomen was opened. The arteriae and venae iliacae werecarefully isolated from the surrounding tissue, and the arterieswere held by threads. A 12-gauge cannula was inserted into thefemoral vein. The arteriae and venae iliacae were clampedabove the holding thread. The arteriae iliacae were lifted bythe thread and opened, and perfusion was started through theinserted cannula. We used 20 ml of 37Â°dissociating medium

for a perfusion of about 1 min. In most cases, it was notpossible to obtain a homogeneous perfusion of all parts ofthe tumor. After perfusion, the tumor was excised and furtherdissociation was performed by shaking as described for normaland regenerating liver.

Cell Yield and Trypan Blue Exclusion. Five aliquots of 0.5ml of the final suspensions were added to 25 ml of the filteredsolution of trypan blue. After 1 hr incubation at 37Â°under

95% O2 plus 5% CO2, cells were counted in a Neubauerhemocytometer. Counting was repeated on 8 separate aliquotsper experiment. The average number of cells per counting was45 Â±5.

Incorporation of L-Leucine-1-14C into Protein. Thesuspensions containing L-leucine-l-14C were incubated at 37Â°

under 95% 02 plus 5% C02. Radioactive protein was isolatedaccording to the method of Mans and Novelli (63, 64).Radioactivity was determined as described previously (75).

NOVEMBER 1972 2569



MÃ¼ller,Schreiber, Kartenbeck, and Schreiber

Electron Microscopy. Samples for the electron microscopywere prepared as described elsewhere (86).

RESULTS

Cell suspensions were prepared by shaking the minced tissuein dissociation medium or by continuous perfusion of the liverwith dissociation medium. Dissociating agents were TPB,EDTA, hyaluronidase, neuraminidase, and collagenase. Totalyield and percentage of unstained cells after 1 hr of incubationin trypan blue are given in Table 1. Two experiments wereperformed for each dissociation technique. In some experiments, when the tissue was dispersed in hyaluronidase pluscollagenase, bovine serum albumin was added to thedissociation medium. No consistent effect of bovine serumalbumin on total yield and proportion of unstained cells wasobserved.

Normal Liver. Unstained cells were obtained only afterdissociation with hyaluronidase plus collagenase. Collagenasecould not be replaced by neuraminidase. When the continuousrecirculating perfusion was used, the proportion of unstainedcells was higher than for the best version of the shakingmethod.

Incorporation of L-leucine-l-14C into protein of liver cells

as a function of time is shown in Chart 1. Cells prepared bycontinuous perfusion incorporated 60 times more L-leucine-

1-14C into protein than cells prepared by shaking in

hyaluronidase plus collagenase or by shaking in TPB.The majority of the cells prepared by continuous perfusion

in situ with hyaluronidase plus collagenase had a well-preserved ultrastructure with an intact plasma membrane,parallel stacks of rough endoplasmic reticulum, and glycogenparticles (Fig. 1). The only difference from the aspect of anormal hepatocyte in tissue was the loss of the polyhedralshape and the very small number of microvilli (Fig. 1, b and c).Broken cells and large intracellular vacuoles (Fig. la) wereseldom observed. In contrast to continuous perfusion, shakingof the minced tissue in TPB yielded swollen cells with adisrupted plasma membrane, a vesiculated endoplasmicreticulum, and disintegrated mitochondria, which had lost mostof their cristae (Fig. 2, a and b). Shaking of the minced tissuein hyaluronidase plus collagenase yielded a homogenate withmany isolated vesicles, pieces of swollen endoplasmicreticulum, inflated or washed out mitochondria (Fig. 36), andbadly preserved nuclei. A few cells were also obtained (Fig.3a). They contained large cytoplasmic vesicles, condensedmitochondria, and a relatively electron-transparent nucleo-plasm. Some cells were partly surrounded by a 2nd membranepresumably originating from adjacent cells (Fig. 3a).

Regenerating Liver. Only enzymatic dispersion was used toobtain single cells from regenerating liver. With the shakingprocedure, 9 to 12 million cells were obtained from l g ofliver. From 10 to 32% of those cells were not stained with

Table 1Preparation of single cells from normal liver and from Morris hepatomas 9121 and 5123tc

Normal liver Morris hepatoma 9121 Morris hepatoma 512 3tc

No.TreatmentCells/g,wet wtX 10"'%

unstainedcells0Cells/g,wet wtX IO'6%

Cells/g,unstained wet wt

cells" X 10~6% unstainedcells"A.

Shaking123456TPB,0.05mMTPB,

0.1mMTPB,

3.0mMTPB,

3.0mM,pH8.6TPB,

3.0mM,PH8.6+glutamate,

5.0mMEDTA,0.2%1.7b0.6b3.50.44.63.73.13.83.61.000000000000.80.74.33.28.39.45.013.26.800000

14.5033.60018.600

7 EDTA, 0.5%

8 EDTA, 0.2% +lysozyme, 0.05%

9 Hyaluronidase, 0.1% +neuraminidase, 5 X 10"

10 Hyaluronidase, 0.1% +collagenase, 0.05%

11 Hyaluronidase, 0.1% +collagenase, 0.05%

0.61.80.30.40.31.70.5

11.07.4

0000000

10.4

3.911.65.6

9.710.7

13.6

73.573.7

B. Continuous perfusion28. T18.06

4.82.3

18.022.5

9.914.8

77.037.3

" Each value is the mean of 8 countings.

Values are obtained from 2 separate experiments.





LUU

Â£20

o.â€¢¿�o

LU

O(EO.

10.75

1.0

0.5

20 Â¿O

MIN INCUBATION

60

Chart 1. Incorporation of L-leucine-l-14C into protein of liver cells.o, perfusion with hyaluronidase + collagenase (left ordinate); a, shakingwith hyaluronidase + collagenase; â€¢¿�,shaking with TPB (right ordinate).

trypan blue. Continuous perfusion yielded 5 to 7 million cellsfrom l g of liver with 28 to 52% unstained cells.

Morris Hepatomas 9121 and 5123tc. It was not possible toestablish continuous recirculation in hepatomas. However,unstained cells from the hepatomas were obtained in the sameyield by the shaking technique as by the continuousrecirculation technique for normal and regenerating liver. Incontrast to normal and regenerating liver, unstained cells werealso observed after treatment with EDTA (Table 1).

Incorporation of L-leucine-l-14C into protein of cells

obtained from Morris hepatoma 5123tc is described as afunction of time in Chart 2. In contrast to normal liver,dissociation of the minced tissue by hyaluronidase pluscollagenase produced cells that incorporated about 300 timesmore L-leucine-l-14C into protein than cells obtained with

TPB.Tumor cells prepared by dissociation with hyaluronidase

plus collagenase showed a well-preserved ultrastructure, whichwas comparable to that of Morris hepatoma cells in situ.Rough endoplasmic reticulum and nuclear envelopes weredilated (Fig. 4, a and b). In cells prepared by TPB, theultrastructure was severely altered (Fig. 5), as described abovefor liver cells (Fig. 2, a and b).

DISCUSSION

We could not confirm that EDTA plus lysozyme wassuperior to hyaluronidase plus collagenase for the preparationof single rat liver cells, as claimed by Hommes et al. (36,37).After dispersion in EDTA with or without lysozyme, all cellsstained with trypan blue. This is in agreement with thereported alteration of the permeability (28, 29, 51, 59, 85).Furthermore, a low rate of respiration in calcium-containingmedia in cells prepared with EDTA plus lysozyme wasdescribed (36). Destruction of the ultrastructure by EDTA

-0.25

20 Â«0

MIN INCUBATION

Chart 2. Incorporation of L-leucine-1-1 4C into protein of tumor cells

prepared from Morris hepatoma 5123tc. o, shaking with hyaluronidase+ collagenase (left ordinate); â€¢¿�,shaking with TPB (right ordinate).

(17, 31, 36, 65, 66) and a loss of enzymes after treatment withlysozyme (19) were also reported.

Like other authors (26, 32, 60), we were not able to obtainunstained cells with the concentrations of TPB published byRappaport and Howze (71â€”73). Our results are in agreementwith the previously reported impairment of protein synthesis(25, 60, 61) and with the observed morphological alteration ofcell organdÃes (26, 31, 60, 66). In mitochondria 0.08 to 0.2mM TPB had an uncoupling effect on respiration and oxidativephosphorylation (87). Impairment of enzyme induction (25,32), leakage of enzymes (26, 81), low endogenous respiration(81), and a lack of the synthesis of lipids (60, 61) aftertreatment with TPB were also described. In liver slices, uptakeof oxygen and retention of potassium ions were remarkablydecreased by 3 mM TPB (67).

Preparation of single cells with hyaluronidase pluscollagenase was also feasible for regenerating rat liver.Furthermore, unstained cells with intact ultrastructure andwith a high rate of protein synthesis could be isolated fromtransplantable hepatomas only with hyaluronidase pluscollagenase.

ACKNOWLEDGMENTS

We are very grateful to Dr. R. Reynolds and Dr. W. W. Franke forcritical discussions and to Dr. H. P. Morris for providing the hepatomas.

REFERENCES

1. Anderson, N. G. The Mass Isolation of Whole Cells from Rat Liver.Science, 117. 627-628,1953.

2. Ap Gwynn, I., Jones, B., Jones, B. M., and Kemp, R. B. A ModifiedPerfusion Technique for Preparing Isolated Liver Cells: AnUltrastructural Study. Cytobios, 7-8: 181-191, 1970.

3. Berry, M. N. Metabolic Properties of Cells Isolated from AdultMouse Liver. J. Cell Biol., 15: 1-8, 1962.

NOVEMBER 1972 2571




4. Berry, M. N. The Action of Fructose on Ethanol Metabolism byIntact Isolated Liver Cells. Biochem. J., 123: 40P, 1971.

5. Berry, M. N. The Action of Pyruvate on Ethanol Oxidation byIntact Isolated Liver Cells. Biochem. J., 123: 41P, 1971.

6. Berry, M. N., and Friend, D. S. High-Yield Preparation of IsolatedRat Liver Parenchyma! Cells. J. Cell Biol., 43: 506-520, 1969.

7. Berry, M. N., and Simpson, F. O. Fine Structure of Cells Isolatedfrom Adult Mouse Liver. J. Cell Biol., /5: 9-17, 1962.

8. Branster, M. V., and Morton, R. K. Isolation of Intact Liver Cells.Nature, 180: 1283-1284, 1957.

9. Burton, D. N., Collins, J. M., and Porter, J. W. Biosynthesis of theFatty Acid Synthetase by Isolated Rat Liver Cells. J. Biol. Chem.,244: 1076-1077, 1969.

10. Capuzzi, D. M., and Margolis, S. Metabolic Studies in Isolated RatLiver Cells. 1. Lipid Synthesis. Lipids, 6: 601-608, 1971.

11. Capuzzi, D. M., and Margolis, S. Metabolic Studies in Isolated RatLiver Cells. II. Biosynthesis of Serum Low Density Lipoproteinsand Its Regulation. Lipids, 6: 609-616, 1971.

12. Capuzzi, D. M., Rothman, V., and Margolis, S. Simplified Methodfor Isolation of Intact Avian and Rat Liver Parenchymal Cells.Biochem. Biophys. Res. Commun., 45: 421-429, 1971.

13. Carr, K. E., Arbuthnott, J. P., Toner, P. G., and Gemmel, C. G.TheFine Structure of Rat Liver Cells in Suspension. Z. Zellforsch., 79:265-271,1967.

14. Casanello, D. E., and Gerschenson, L. E. Some Morphological andBiochemical Characteristics of Isolated Rat Liver Cells Dissociatedwith Sodium Tetraphenylboron and Cultured in Suspension. Exptl.Cell Res., 59: 283-290, 1970.

15. Castagna, M., and Chauveau, J. Dispersion du Tissu HÃ©patiqueÃ l'Ã‰tatde Cellules IsolÃ©es.Compt. Rend., 257: 969-972, 1963.

16. Castagna, M., and Chauveau, J. CaractÃ©ristique Chimiques etEnzymatiques des HÃ©patocytes Isoles. Arch. Sci. Physiol., 22:229-246, 1968.

17. Coman, D. R. Cellular Adhesiveness in Relation to the Invasivenessof Cancer: Electron Microscopy of Liver Perfused with a ChelatingAgent. Cancer Res., 14: 519-521, 1954.

18. Cooper, B., and Margolis, S. Inhibition of Lipid Synthesis inIsolated Rat HÃ©patocytesby Serum Lipoproteins. J. Lipid Res.,12: 731-739, 1971.

19. Crisp, D. M., and Pogson, C. I. Glycolytic and GluconeogenicEnzyme Activities in Parenchymal and Non-parenchymal Cellsfrom Mouse Liver. Biochem. J., 726: 1009-1023, 1972.

20. Curtis, J. C., Lawner, P., and Minasian, D. Effect of theConcentration of Collagenase upon the Dispersion of ParenchymalCells of Mouse Liver. J. Cell Biol., 39: 30a-31a, 1968.

21. Dickson, J. A. The Uptake of Non-metabolizable Amino Acids asan Index of Cell Viability in Vitro. Exptl. Cell Res., 61: 235-245,1970.

22. Dickson, J. A. Metabolism of Adult Rat Liver Cells in Filter-WellCulture. Exptl. Cell Res., 64: 17-28, 1971.

23. Exton, J. H. Metabolism of Rat-Liver Cell Suspensions. 1. GeneralProperties of Isolated Cells and Occurrence of the Citric AcidCycle. Biochem. J., 92: 457-467, 1964.

24. Felts, J. M., and Berry, M. N. The Metabolism of Free Fatty Acidsand Chylomicron TriglycÃ©rideFatty Acids by Isolated Rat LiverCells. Biochim. Biophys. Acta, 231: 1-7, 1971.

25. Fricdmann, T., and Epstein, C. J. The Incorporation of[3H)-Leucine into Protein by Tetraphenylboron- andCitrate-Dispersed Rat Liver Parenchymal Cells. Biochim. Biophys.Acta, 138: 622-624, 1967.

26. Gallai-Hatchard, J. J., and Gray, G. M. A Method of Obtaining aSuspension of Intact Parenchymal Cells from Adult Rat Liver. J.Cell Sci.,Â«:73-86, 1971.

27. Gerschenson, L. E., and Casanello, D. Metabolism of Rat LiverCells Cultured in Suspension: Insulin and Glucagon Effects on

Glycogen Level. Biochem. Biophys. Res. Commun., 33: 584-589,1968.

28. Gibbons, J. L., and Rienits, K. G. Diphosphopyridine Nucleotideand the Respiration of Rat-liver-cell Suspensions. Biochim.Biophys. Acta, 47: 395-397, 1961.

29. GÃ¼nther,T., and Goecke, C. Ã„nderung der PermeabilitÃ¤tbei derIsolierung von Zellen. Z. Naturforsch.,21b: 1171-1174, 1966.

30. Hanks, J. H., and Wallace, R. E. Relation of Oxygen andTemperature in the Preservation of Tissues by Refrigeration. Proc.Soc. Exptl. Biol. Med., 71: 196-200, 1949.

31. Harris, C. C., and Leone, C. A. Some Effects of EDTA andTetraphenylboron on the Ultrastructure of Mitochondria in MouseLiver Cells. J. Cell Biol., 28: 405-408, 1966.

32. Haung, Y. L., and Ebner, K. E. Induction of TyrosineAminotransferase in Isolated Liver Cells. Biochim. Biophys. Acta,191: 161-163, 1969.

33. Hayek, D. H., and Tipton, S. R. Respiratory Activity andMaintenance of Cell Suspensions of Rat Liver. J. Cell Biol., 29:405-409, 1966.

34. Henley, K. S., Sorensen, O., and Pollard, H. M. Some EnzymaticProperties of Suspensions of Parenchymatous Liver Cells. Nature,184: 1400, 1959.

35. Higgins, G. M., and Anderson, R. M. Experimental Pathology ofthe Liver. I. Restoration of the Liver of the White Rat FollowingPartial Surgical Removal. A.M.A. Arch. Pathol., 12: 186-202,1931.

36. Hommes, F. A., Draisina, M. L, and Molenaar, I. Preparation andSome Properties of Isolated Rat Liver Cells. Biochim. Biophys.Acta, 222: 361-371, 1970.

37. Hommes, F. A., Oudman-Richters, A. R., and Molenaar, I. ThePreparation of Isolated Fetal Rat Liver Cells. Biochim. Biophys.Acta, 244: 191-199, 1971.

38. Howard, R. B., Christensen, A. K., Gibbs, F. A., and Pesch, L. A.The Enzymatic Preparation of Isolated Intact Parenchymal Cellsfrom Rat Liver. J. Cell Biol., 35: 675-684, 1967.

39. Howard, R. B., and Green, C. The Metabolism of Liver CellSuspensions. Biochem. J., 96: 33P, 1965.

40. Howard, R. B., and Pesch, L. A. Respiratory Activity of Intact,Isolated Parenchymal Cells from Rat Liver. J. Biol. Chem., 243:3105-3109, 1968.

41. Ichihara, A., Adachi, K., Daikuhara, Y., and Takeda, Y. TheBiochemistry of Animal Cells. II. Lipid Biosynthesis in DispersedRat Liver Cells. J. Biochem., 57: 696-705, 1965.

42. Ichihara, A., Koyama, E., and Takeda, Y. The Biochemistry ofAnimal Cells. IV. Biosynthesis of Cholesterol and Its Regulation inDispersed Rat Liver Cells. J. Biochem.,58: 480-486, 1965.

43. Ichihara, A., Tanioka, H., and Takeda, Y. Respiratory Control ofDispersed Rat-Liver Cells. Biochim. Biophys. Acta, 97: 1-8, 1965.

44. Ingebretsen, W. R., Jr., and Wagle, S. R. A Rapid Method for theIsolation of Large Quantities of Rat Liver Parenchymal Cells withHigh Anabolic Rates. Biochem. Biophys. Res. Commun., 47:403-410, 1972.

45. lype, P. T., and Bhargava, P. M. The Respiration of IsolatedRat-Hepatic Cells in Suspension. Biochem. J., 94: 284-288, 1965.

46. Jacob, S. T., and Bhargava, P. M. A New Method for thePreparation of Liver Cell Suspensions. Exptl. Cell Res., 27:453-467, 1962.

47. Jacob, S. T., and Bhargava, P. M. Metabolism of Tissue Cells inSuspension. Synthesis of Ribonucleic Acid by Liver Cells inSuspension. Biochem. J., 95: 568-576, 1965.

48. Jacob, S. T., and Bhargava, P. M. Effect of Chloramphenicol onRibonucleic Acid Synthesis in Liver Cells in Suspension. Biochem.J., 97. 67-73, 1965.

49. Jezyk, P. F., and Liberti, J. P. Metabolic Activities of Mechanically





and Enzymatically Prepared Rat Liver Cells. Arch. Biochem.Biophys., Â¡34:442-449, 1969.

50. Jungalwala, F. B., and Dawson, R. M. C. Phospholipid Synthesisand Exchange in Isolated Liver Cells. Biochem. J., 117: 481-490,1970.

51. Kalant, H., and Miyata, R. Metabolic Alterations in TissuesPerfused with Decalcifying Agents. Biochem. J., 86: 336-344,1963.

52. Kalant, H., and Young, F. G. Metabolic Behaviour of Isolated Liverand Kidney Cells. Nature, 179: 816-817, 1957.

53. Kaltenbach, J. P. Preparation of Suspensions of Intact Liver Cells.Federation Proc., 11: 237-238, 1952.

54. Kaltenbach, J. P. The Preparation and Utilization of Whole CellSuspensions Obtained from Solid Mammalian Tissues. Exptl. CellRes., 7: 568-571, 1954.

55. KrÃ¼ger,W., and Schnitzler, S. /Â«-K/Vo-Wachstumsverhalten vonZajdela-Asciteshepatomzellen nach in-Vitro-Behandlung mitTrypsin und Pronase. Z. Naturforsch., 26b: 169-170, 1971.

56. Lata, G. F., and Reinertson, J. Cholesterol Synthesis by IsolatedRat Liver Cells. Nature, 779: 47-48, 1957.

57. Laws, J. O., and Stickland, L. H. Metabolism of Isolated LiverCells. Nature, / 78: 309-310, 1956.

58. Laws, J. O., and Stickland, L. H. The Adhesion of Liver Cells.Exptl. Cell Res., 24: 240-254, 1961.

59. Leeson, T. S., and Kalant, H. Effects of m Vivo DÃ©calcificationonUltrastructure of Adult Rat Liver. J. Biophys. Biochem. Cytol., 10:95-104, 1961.

60. Lipson, L. G., Capuzzi, D. M., and Margolis, S. Effect of Method ofCell Isolation on the Metabolic Activity of Isolated Rat Liver Cells.J. Cell Sci.. 10: 167-179,1972.

61. Lipson, L. G., Margolis, S., and Capuzzi, D. M. Comparison ofAcetate and Amino Acid Incorporation in Rat Liver Cells Isolatedby Three Techniques. Federation Proc., 28: 366, 1969.

62. Longmuir, I. S., and ap Rees, W. Preparation of Cell Suspensionsfrom Rat Livers. Nature, 777: 997, 1956.

63. Mans, R. J., and Novelli, G. D. A Convenient, Rapid and SensitiveMethod for Measuring the Incorporation of Radioactive AminoAcids into Protein. Biochem. Biophys. Res. Commun., 3:540-543, 1960.

64. Mans, R. J., and Novelli, G. D. Measurement of the Incorporationof Radioactive Amino Acids into Protein by a Filter-Paper DiskMethod. Arch. Biochem. Biophys., 94: 48-53, 1961.

65. Mills, D. Morphology of Trypsin Separated Rat Liver Cells.Federation Proc., 26: 576, 1967.

66. Mills, D. M., and Zucker-Franklin, D. Electron Microscopic Studyof Isolated Kupffer Cells. Am. J. Pathol.,54: 147-155, 1969.

67. Murthy, L., and Petering, H. G. A Comparison of SomeBiochemical Properties of Dissociated Liver Cells and of Slices.Pioc. Soc. Exptl. Biol. Med., 132: 931-935, 1969.

68. Ontko, J. A. Chylomicron, Free Fatty Acid and Ketone BodyMetabolism of Isolated Liver Cells and Liver Homogenates.Biochim. Biophys. Acta, 137: 13-22, 1967.

69. Pertoft, H. The Separation of Rat Liver Cells in ColloidalSilica-Polyethylene Glycol Gradients. Exptl. Cell Res., 57:338-350, 1969.

70. Poste, G. Tissue Dissociation with Proteolytic Enzymes. Exptl. CellRes., 65: 359-367, 1971.

71. Rappaport, C., and Howze, G. B. Dissociation of Adult MouseLiver by Sodium Tetraphenylboron, a Potassium ComplexingAgent. Proc. Soc. Exptl. Biol. Med., 121: 1010-1016, 1966.

72. Rappaport, C., and Howze, G. B. Further Studies on theDissociation of Adult Mouse Tissue. Proc. Soc. Exptl. Biol. Med.,121: 1016-1021, 1966. p

73. Rappaport, C., and Howze, G. B. Effect of Temperature *on

Dissociation of Adult Mouse Liver with Sodium Tetraphenylboron.Proc. Soc. Exptl. Biol. Med., 121: 1022-1025, 1966.

74. Rotermund, H.-M., Schreiber, G., Maeno, H., Weinssen, U., andWeigand, K. The Ratio of Albumin Synthesis to Total ProteinSynthesis in Normal Rat Liver, in Host Liver, and in MorrisHepatoma 9121. Cancer Res., 30: 2139-2146, 1970.

75. Schreiber, G., Urban, J., ZÃ¤hringer,J., Reutter, W., and Frosch, U.The Secretion of Serum Protein and the Synthesis of Albumin andTotal Protein in Regenerating Rat Liver. J. Biol. Chem., 246:4531-4538, 1971.

76. Segard, E., Montreuil, J. Colbeau, A., Mangez, C., Dupont, A.,DÃ©maille,A., and Driessens, J. Nouveau ProcÃ©dÃ©Original dePrÃ©parationde Cellules Vivantes par Dissociation du Foie de Rat.Experientia, 20: 467-469, 1964.

77. Shanmugam, G., and Bhargava, P. M. The Uptake of HomologousRibonucleic Acid by Rat-Liver Parenchyma! Cells in Suspension.Biochem. J., 99: 297-307, 1966.

78. Shanmugam, G., and Bhargava, P. M. The Effect of Actinomycin Don the Synthesis of Ribonucleic Acid and Protein in the Rat LiverParenchyma! Cells in Suspension and Liver Slices. Biochem. J.,108: 741-748, 1968.

79. Shanmugam, G., and Bhargava, P. M. Uptake of Escherichia ColiRNA by Rat Liver Cells in Suspension. Indian J. Biochem., 6:64-70, 1969.

80. Stacpoole, P. W., and Berry, M. N. DiisopropylammoniumDichloroacetate: Effect on Gluconeogenesis in Isolated Rat LiverCells. Federation Proc., 30: 329, 1971.

81. Suzangar, M., and Dickson, J. A. Biochemical Studies on CellsIsolated from Adult Rat Liver. Exptl. Cell Res., 63: 353-364,1970.

82. Takeda, Y., Ichfliara, A., Tanioka, H., and Inoue, H. TheBiochemistry of Animal Cells. I. The Effect of Corticosteroids onLeakage of Enzymes from Dispersed Rat Liver Cells. J. Biol.Chem., 239: 3590-3596, 1964.

83. Terranova, T., Baldi, S., Galeotti, T., and Neri, G. MetabolicControl in Isolated Rat Liver Cells. Epatologia, 13: 935-946,1967.

84. Thimmappayya, B., Ramachandra Reddy, R., and Bhargava, P. M.Preparation of Kidney Cell Suspensions and Respiration of Kidneyand Liver Cell Suspensions. Exptl. Cell Res., 63: 333-340, 1970.

85. Tsai, C. M., Best, N., and Ebner, K. E. Retention of LÃ¡clateDehydrogenase, Cortisol Binding, and Lack of Enzyme Inductionin Rat Liver Cell Suspensions. Exptl. Cell Res., 44: 332-340,1966.

86. Urban, J., Kartenbeck, J., Zimber, P., Timko, J., Lesch, R., andSchreiber, G. Increase of Extravascular Albumin Pool and theIntracellular Accumulation of Vesicles in Transplanted MorrisHepatoma 9121. Cancer Res., 32: 1971-1977, 1972.

87. Utsumi, K., and Packer, L. Uncoupling of Energy TransferReactions in Mitochondria by Tetraphenyl Boron. Arch. Biochem.Biophys., 722: 509-515, 1967.

88. Waymouth, C. Rapid Proliferation of Sublines of NCTC Clone 929(Strain L) Mouse Cells in a Simple Chemically Defined Medium(MB 752/1). J. Nati. Cancer Inst., 22: 1003-1017, 1959.

89. Weigand, K., MÃ¼ller,M., Urban, J., and Schreiber, G. IntactEndoplasmic Reticulum and Albumin Synthesis in Rat Liver CellSuspensions. Exptl. Cell Res., 67: 27-32, 1971.

90. Weiss, L. Studies on Cell Deformability. II. Effects of SomeProteolytic Enzymes. J. Cell Biol., 30: 39-43, 1966.

91. Yamada, T., and Ambrose, E. J. Preparation of Cell Suspensionsfrom Tissues Using Collagenase and Hyaluronidase, Especially forMeasuring the Electrical Charges of Surface Membranes ofMammalian Cells. Exptl. Cell Res., 44: 634-636, 1966.

92. Zimmermann, M., Devlin, T. M., and Pruss, M. P. AnaerobicGlycolysis of Dispersed Cell Suspensions from Normal andMalignant Tissues. Nature, 185: 315-316, 1960.

NOVEMBER 1972 2573




m '**&&&$Â£''^ Ã„'>*Â¿fÃ t&j$&-' â€¢¿�'â€¢<*:" â€¢¿�i r^7i *

^^â€¢'^^â€¢^^-. C- ^l> ' -

^fe^^^tÃ³feÂ¿-0t*Ã„-V:^V<- ;^%*V

S, ^^Â«S^HfcL^wSvÂ»

v:^Â¿aeÂ£^ :-;Ã„5

'-,SÃ•)Ã•^AS;;-V^

le





"* J*Z*SÂ¿ ffirSrar \--#" o - . , wvâ€¢"....

â€¢¿�'at,-'-^-rx-.Ir- r ' -^ER-T Â£, J*-*

Bsi^y'Â«-- Â«^ -^TE&gÃ¤ < â€¢¿�'$Â£;â€¢!&â€¢,.''â€¢'}

LM Â«*RÂ«&*?Â«*^ !rÂ«

Ã^|r:

Figs. 1 to 5. Electron micrographs of glutaraldehyde-fixed cells postfixed with osmium tetroxide. Sections were stained with uranyl acetate andlead citrate. ER, endoplasmic reticulum; sER, smooth ER; rER, rough ER; G, glycogen;/, junctional complex; L, lipid droplet\Ly,, lysosome;M,mitochondrion;MÃŸ, microbody Â¡A',nucleus; PC, perinuclear cisterna.

Fig. 1. Cells prepared from normal rat liver by continuous perfusion with hyaluronidase plus collagenase. a,, single isolated cell. X 7,000.b, parallel stacks of rough endoplasmic reticulum, vesicles of smooth endoplasmic reticulum, and glycogen particles (arrows). X 30,000.c, well-preserved cell organelles and an intact plasma membrane. X 22,000. d to /, normal (d) and condensed (e) mitochondria and both types in

immediate vicinity (/)-X 30,000; X 18,000; X 28,000, respectively.Fig. 2. Parts of cells prepared from normal rat liver by shaking of the minced tissue in TPB. All cell organelles are swollen or disintegrated. The

plasma membrane is disrupted at many sites (arrows). In b, a part of a junctional complex is visible, a, X 7,000; b, X 33,000.Fig. 3. Parts of cells prepared from normal rat liver by shaking of the minced tissue in hyaluronidase plus collagenase. a, condensed

mitochondria, few single cisternae of rough endoplasmic reticulum, and electron-transparent nucleoplasm. The cell is partly surrounded by a 2ndmembrane (arrows) presumably originating from adjacent cells. X 18,000. b, homogenate-like aspect. Most mitochondria appear inflated or leakedout. Irregularly shaped cytoplasmic vesicles, lipid droplets, and single cisternae of endoplasmic reticulum can be seen. X 16,000.

NOVEMBER 1972 2575




* *

l$p^$M ' *

4 a

** J

4b

'vfc^rÂ¿V''" ^ J^1*.

. . â€¢¿�- â€¢¿�~<Â«i"'5 -'

; prru

Fig. 4. CeUs prepared from Morris hepatoma 5123tc by shaking of the minced tissue in hyaluronidase plus collagenase. a, single isolated cell.X 9,000. b, section of a. Well-preserved cell organelles, intact plasma membrane with microvilli-like structures, and many free ribosomes. The singlecisternae of the endoplasmic reticulum and the nuclear space are dilated. X 20,000.

Fig. 5. Part of a cell prepared from Morris hepatoma 5123tc by shaking of the minced tissue in TPB. Swollen and disrupted organelles.Discontinuity in the plasma membrane (arrow). X 9,000.




1972;32:2568-2576. Cancer Res Matthias Müller, Margot Schreiber, Jürgen Kartenbeck, et al. Regenerating Liver, and Morris Hepatomas 9121 and 5123tcPreparation of Single-Cell Suspensions from Normal Liver,

Updated version

http://cancerres.aacrjournals.org/content/32/11/2568

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/32/11/2568To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



preparation of single-cell suspensions from normal liver ... · hanks' solution (30)...

Documents