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Differential susceptibility of epithelial cells and fibroblasts of human skin to freeze injury
CRYO|~I'OLOGY
VOI. 5, No. 4, 1969
DIFFERENTIAL SUSCEPTIBILITY OF EPITHELIAL CELLS AND FIBROBLASTS OF HUMAN SKIN TO FREEZE INJURY R e p o r t o n an I m p r o v e d M e t h o d f o r S t o r a g e ot~ H u n m n S k i n at - 1 9 6 ° C ~
BALU It. A-THREYA, E. L. GRIMES, ttERNDON B. LEHR, ARTI=tUI-¢ E. GRE~ENE, AND LEWIS L. CORIELL
I.a~titute /or Medical Research, Camden, New Jersey, and HarH.~on Department el Surgical Research, University o/Pennsylvania School o] Medicine, Philadelphia, Pen'nsylvania years old. Skin was cut into ,approximately 1cm~square pieces. The pieces were placed in a medium in which they were to be frozen. At'ter 1 hr, ,the pieces were placed in sterile finger cots containing 3 ira1 of the corresponding medium. The finger, cots were then placed inside a sterile surgics,l glove and frozen in a Linde.BF-lfreezer at 1 to 2°C per man or in tl~e vapor phase of a liquid nitrogen tank at 7 to 8°C per mira When t h e temperature tions, stnnula,ted u s to study the effects of reached --100°C, the gloves were lowered into various •parsmeters of pretreatment and 5quid nitrogen and left there until ready for freezing/onyiabifity of epithelial and fibro- thawing. Thawing I was done. by agit'~ting the blastic elements of human skin by a "tissue finger cots rapidly ,in. water kept at 38°C. To test for viabiliW, eight to 10 small pieces culture' method. Prefimina:iT work indicated that, when (1 to 2 ram) of skin were placed in plasma fresh rat s k i n wasplaced in ~k plasma clot clot in plastic Petri dishes, covered with 5 ml ct~ture,"epithetial cellsgrew ou~ first, f'ollowed of Eagle's medium in Ea.rle's balanced salt 3 to :6: days later by a ring of fibroblasts at the solution with 10% inactivated newborn calf periphery.~However, when the same pieces were serum (EE medium), and maintained at 37°C treated,~vith" 10% glycerol a t 4 ° c for 1 hr, in a C0.~ incubator. Plates were read every fi.ozdn~(:ith~Wed,:and cultured, fibroblast~s grew 3rd day or more frequently, and the nature of out at about:.the same ,txme but ep~tnelml outgrowth was noted. The outgrowth was graded from 1+ to 4+. When the edge of a ceils we?e:del~e4 or absent. Tn/thiS rel~o~t:}'evidence is presented to show piece of skin was focused under 100X of an their: freez~ng"of:~fUll thickness adult human inverted: microscope~ witli the edge of skin stdn at. ~1967C ::results :in :more damage to visible in one~6orner, the presence of only ep~tSeliaLeellS::than.:tOtheifibroblasts and that seven to eight cells or fewer was rated as #itheiial ~eJemeli~ of :skin. are better preserved "occasional outgrowth/' When the skin ~howed iii: Skiii t)retreated 'with a medium containing outgrowth of cells reaching 25% of the .area visible under the microscope, it was graded 1+. 20' i030% glycer01 for ~It0 2hrs at 4°c. When the cells covered .~A)%of the area, it was 2 + ; i f the cells covered 75% of the area, it !-~V~a.TERIAL$ AND ~ E T H O D S was 3 + ; and if the cells covered the entire Skin was obtained from patients :undergoing elective su~geo-.:M6s~: of. tile pieces w'ere :from area visible, it Wa~s4+. the ~ abdomen"of patieiits ~i~)et(ve6n30 and 40 RESULTS
~ o l ~ et al.'° observed that rat skin soaked in 10% glycerol or 10% dimethyl sulfoxide (DMSO) for 2 hrs at 24°C and cooled to 4°C before, freezing decreased i n size by about 25% When, grafted back to the same animal, whereas nonfrozen controls showed no shrinkage~ when huma~ skin stored at ,-196°C in a medium Containing 10% glycerol was placed on piasma :clot culture, there was no outgrowth of t
:Before ~freezing, explants of human skin Receiyed August ,14i 1968. * Tl~is:'inves~igati~h:,WaS;Supportedin par~ bY a showed/an initial outgrowth of epithelial cells gran~ (fr~m,the 30hn~A.-'Harfford.Fo~mdatSon and as early as 3 days in culture, and fibroblasts in par t b y P u b l i c Health Serkice Research Gran~ grew out by about the 6th day. The final CA-0~!953--09 from the ~Na~mnal " " ' ' Cancer Institute. 262
DIFFERENTIAL SUSCEPTIBILITY SKIN VI~'~iLITY
203
STUDIES EPffHELJAL- UNFROZEN 4-t EPITHELIAL-FROZEN "RBROBLA~- UNFR(IZEN RBROBLAST- FRffZEN
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DAYS AFTER CULTURE
Fio. 1. Comparison of tissue culture characteristics of fresh human skin with those of a matching piece frozen after pretrcatment with 10% glycerol at 4°C for 1 hr. Note that in unfrozen skin epithelial cells ap!sear very early and make a full sheet whereas fibroblasts appear later. In matching pieces of frozen-thawed skin fibroblasts appear iust as in unfrozen skin s~ad make a better sheet than in unfrozen skin. Epithelial cells appear after 12 days and make a smaller sheet. (Grades 1 to 4 are described in text.) outgrowth of epithehal cells was usually 4+, and that of fibroblast, usually 2+. However, when human skin was treated with 10% glycerol or 10¢o DMSO for 1 to 4 hr,s at 4°C and then frozen, thaived, and cultured, the epithelial cells did not come out until about t0 to 12 days after culture. Fibrbblasts started growing by the 6th day of culture similar to unfrozen sldn,-and the final outgrowth of fibroblasts was generally 3+ (Figs. 1, 2, and
3). Duration o] pretreatment. Skin was cut into 28 pieces and left in medium containing either 10% glycerol or 10% DMSO for V2, 1, 2, 4, 8, and 24 hrs at 4°C before freezing. Skin cultures were prepared before and after freezing. Pieces of skin pretreated in glycerol or DMSO for less than 1 hr before freezing showed outgrowth of cells after 12 days i n culture. When the skin w a s pretreated with 10% glycerol or 10% DMSO for 1 to 4 hrs before freezing, there was outgrowth of cells within 6 days. Skin. left in glycerol or DMSO for 8 and 24 hrs before freezing grew out in 8 days. No difference could be demonstrated between the crYoprotective effects of 10% glycerol and 10% DMSO by these techniques. Temperature of pretreatment. Pretrea/mcnt of the skin with 10% glycerol for 1 hr at
4~C was compared with pretreatment at 370C to determine whether glycerol might more easily penetrate the layers of the skin at 37°C and hence protect the epithelial cells. The results sununarized in Table 1 show t h a t there was no better, survival of cells exposed to glycerol at 37°C.
Rage of /reezing on viability of epithelial cells. The effect of rate of freezing on viability was tested as follows. A piece of skin was cut into two halves. One half was frozen i n 10% glycerol at the rate of 7 to 8°C per rain. The other half was frozen rapidly at the rate of 125°C per min. When thawed and cultured; both pieces showed similar outgrowth of cells and .the fibroblasts appeared 3 to 4 days before tile epithelial cells as i n earlier experiments. tIyaluronidase ]or pretreatment. To ,facilitate the- penetration of t h e tissues by : glycerol, hyalur0nidase was , added to m e d i u m containing 10% glycerol. The skin biopsies were ex~ posed ,t° these, m e d i a Containing 10 unitS:,per ml and 50 traits per ml of hyaluronidase at 4°C for 1 hr, before freezing'. Under o~r,:ex,perimental condit, ion; ~he delay ,!n onset ',of growth ,,of epithelial cells: Still,-per~isted, 'irre,spective of t h e presence or absence of hyaluronidase.
264:
ATHREYA ET A L.
l,'~a. 2. Epithelial cell sheet from plasma clot culture of unfrozen human skin.
Varying concentrations of glycerol and epithelial viability. Varying concentrations of glycerol were tested by immersing pieces of skin in E E medium cont.qining 0 to 50% glycerol for 1 hr at 4°C. A small portion of each of these pieces was used for immediate culture. The remaining portion was frozen at the rate of 7 to 8°C per rain. When thawed and cultured, skin frozen in 20 to 30% glycerol showed the most consistent viability (Figs. 4, 5, and 6). Epithelial cells consistently grew out earlier than fibroblasts and formed a riffler sheet after pretreatment with 20 and 30% glycerol m~d eve~ with 40 and 50% glycerol, occasionally. ~'nese experiments were repeated with 20% glycerol at 4°C to detemdne the optimal treatment time before freezing. The results shown in Table 2 indicate the optimal time to be between 1 and 2 hrs as shown by better survival of epithelial cells.
Transplantation of frozen skin. In order to compare the ability of frozen-thawed skin processed by this improved method to grow in tissue culture with its ability to function as viable graft, the following experiment was done. A piece of skin 2 inches by 1 inch was removed from the ventral surface of 15250-g Amsterdam rats and cut into two halves. One half was treated with E E meclium containing 10% glycerol for 2 hrs at 4°C and frozen in glass tubes in 5 mI of the same medium in a Dry Ice chea at --70°C. The other half was immersedin E E medium containing 20% glycerol for 1 hr at 4°C and frozen in the vapor phase of liquid nitrogen. Both pieces were thawed after 24 hrs by agitating in water at 37°C and transplanted to the dorsum of the original donor rat. The grafts were measured on day 0 and on day 28. Plnsma clot cultures of both halves of skin were done before and
DIFFERENTIAL SUSCEPTIBILITY
,.t)o
Fro. 3. Plasma clot culture of human skin (same as in :Fig. 2) but after freezing and thawing. Prctreatment with 10% glycerol at 4°C for 1 hr. Note only fibroblasts. after freezing and were analyzed for outgrowth of epithelial cells and fibroblasts, the day of appearance, and the extent of growth. In all the cultures of sldn before freezing, epigleIitfl cells grew earlier than fibroblasts and made full sheets earlier. This pattern was seen also in skin frozen in 20% glycerol at -196°C. Ilowever, matching pieces of skin frozen in 10./o glycerol at - 7 0 ° C showed early outgrowth of epithelial cells in 50% of the cases, tile fibroblasts growing out earlier in tl~e others (Fig. 7). All of the grafts took well, and we could not confirm the earlier finding of graft shrinkage. f, t
TABLE 1 ~ F F E C ' r OF ])RE'rltEATMENT OF SKIN IN ~IEOIU~t
4°C .,,','u ,~'r 17°C (POS'rm~EZE CVLTm~ES)
COXT.,,IxlXG 10% GW,'CnROL
~. pedment No.
of"
Onset of
0dsct
oi Fibreblasts
o£ Epithellal Cel!
Final Type
E:~tent of Growth
F 1,2. F
4E, 4F
I
1
4 87
2
4 B7
3
-i B7
I)lsc~'ssIox Assay of viability of skin after any storage procedure is ~ difficult problem. Lerggren and Lehr,' Flatt7 ]3illingham and Medawar; ~ Ash-
Te.mperature
.vc
14 11
6
6
14 11
17
I E, F i E, F i E, F 10 E, F t
* For explanation of s:~m~bols see text.
l+*
4E, 4F 4E, .IF 4E, F4 4E: 4F
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EPITHELIAL CELLB FIBROBLASTS
40
50
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0 ~o
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I0
20
30
PERCENTAGE OF GLYCEROL IN THE MEDIUM
Fro. 4. Effects .of 'various concentrations of glycerol in the, pretreatment medium o n the viability of epithehal, cells and fibroblasts (summal'y of 1Oexperiments). Note that, until the glycerol concentration is increased to 20%, fibroblasts always appear earlier than epithelial cells. (In unfr0zen skin, epithelial cells alwuys appear earlier.) FREO,U]@IIC~/"OF ONSI~I' OF OUTGROWTHSOF FIB~OILLASTS ~ CELLS ~
A~'~'D~,P.ITRELI.AL
BFJ~RE DA'I 6 FROM ~I~OZEN-TRAW~ HURAN SKIN
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3O
4O
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P~CE~IT OF C~LI~OL IN THE Y2~IUM
Fm. 5~ Effects of-yangus:concentrations of glycerol in t h e pretreatment medium on the viabi!i~.Y:of,: epithelial: ce!Is !and~ fibrobl~ts: .In order to obtain, condition :.under which epithelial ::cells Can:appear earlier than -6 (lays in tisSue Cultare,: t.lii; glycerol concentration has to goOver20%. 266
I)IFI, EREN TIAL St,,SCLI TIBILI r~2
2G7
~ORRELATIOI~ BETWEEN PERGE2CFA~EOF GLYCEROL IN THE PRETRF.ATHENT&RI} FREEZE NEDIIJH AND SIJCCESSFUL RI~OV~t¥ OF FIBROBLA~TS Ah'D EPITHE1/IAL CELL~ FROH FROZEN-THA~ HUHAN SKIR FIBROI~L~TS
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10
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50
PF/ICEI~',.~GE OF GLYCI~IOL IN THE N~.~IUH
FI¢;. 6. Effects of various concen.tm~_ons of.glycerol in the pretreat,ment medium on the successful outgrowth of epithelial cells and fibrob!astS'on or about~ the 18th day of culture, In this graph, every piece of skin which showed an outgrowth of epithelial cells or fibroblasts was graded as positive, irrespective of the number of days it took for them to .first appear in culture. (In cultures of Unfrozen skin, there is ofltgrowth of epithelial cells and fibroblasts in every instance.) After fl-eezing and thawing the optimal recovery" of both types of cells is 90% when pretreated with a medium containing 20 to 30% glycerol. qhkBl.,l~ 2 I~FFEC'I'S OF ]~ I~L [I,l:,Aq M]~IN 1. OF ]I:IUMAN ~ K I N W I T H
[20%'GLYCEROL a'r-4°C rOit \:AllYINGDURATIONS OF T I M g BI,3FORI~. F . R E E Z I N G
Duration of Pre trea tmen t
Ceil T ~ e to Appear Early in Tissue Culture ~
No, o{ Experiment
Epithelial {Fibroblasts
%
(. 's)
5
0 40
5
40
5 2 2
20
5
1 2 4 8 24
r
100 80 100 ,
100
0
0
80
,
100
* In culfures of .unfrozen skin epithelial cells appear first, in every instance, ley and.Bordley,~ and. Baxter and, Entin~have used .the microscopic appearance of t h e graft for establishing criteria for survival. Billingiaam: a n d Medawar~ established the
following criteri~ for evidence of survival of skin grafts: " ( a ) ~kpI)earanCe of an. mmult~s-:of migratory outgrowth f r o I n t h e , origina! graft, margin over ithe surrounding raw area.... (b) SUr~dval of epidermal melanoblasts..(c)..Preservation of fine, fibrous. orgamsatlon., " ' """ " Using these criteria, -they found-slow freezing foll0wedby rapid thawing was best: for rabbit skin and found glycerol concentrations between"I0-a,nd 3 0 % asl being effective- in reducing ,freeze;ir/2 jury. They i'otmd that whole skin.su'rvivedex: p o s u r e . a t 0°C to" 98.t% ,glycerol-for.. ' -" :8 -lira a n d to 8 0 % .gtyeero! f o r 2 hrs; whereas: a suspension of epithelial cells showed damage.. ag these concentrations of glYeeroI after 35 .ram. In oar studies;...Skin Soak(id :'in:.lOO%:glyeero1 for :1 h r : at.. 4? C. felt" like .:a..piece,"iof CardbOard bu~"sti!Fshowe d ..outgrOwth.. of ePithetiai cells and fibroblasts,bef0re ~,md afterfreeZing. Bminghm :and"J,eynotde: fised i-:pure. :, epidermal 'sheets ~and suSpensi0n'.:of.iepidielihi' ceiis to cover., areas :of skin,.Ioss':in rabN,ts:":The',; '
.
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268
ATtIREYA ET AL.
vivM of skin. It will test not only for cell survival but also for the ability of skin to function as an organized tissue. Skin need not be viable to be effective as a i00 temporary graft and, therefore, freeze-thaw studies of human skin evahmted solely on the gross appearance are not a final criterion of o 75 viability as discussed by Grfilo and h , Khann. ' In our own studies we have shown that four pieces cf human skin from the skin bank 5o of the Hospital of the University of Pennsylo vania showed no outgrowth of cells in tissue culture, although they had functioned ade25 quately as grafts. Tissue culture assay of frozen skin is a C~ simple in vitro assay. :Tile conditions under which the cells have to grow are less favorable F 18RO BL AS TS EPITHELIAL but perhaps more reproducible than when Early Outgrowth grafted onto an animal. In tissue culture, of Cell Types Before Day 3 identification of cells as epithelial cells or Fro. 7. Note that, when stored in 20% glycerol fibroblasts is usually easy. Using electron microscopic study of skTn in plasma, clot culat --196':C, epithelial cells grow o u t e a r l i e r than fibrobtasts, and before day 3 in 90% of samples. tures Friedmar~-t.(ien et al.'~ concluded that, in This is similar to the unfrozen skin. Epithelial the immediate vicinity of the explant, outcells appear earlier in only 50% of samples frozen at --70°C ,xfter pretreat.menl; in a medium con- growfll of epithelioid cells does not necessarily mean absence of fibrob]asts. A]so, in ti~ae taining I0% glycerol. culture of skin a cut sweat gland duct-,may gave good, temporary protection, but the grafts proliferate and show a good-outgrowth of separated from the underlying tissue by about epithelial cells and give false resultsY The results of this study shm~ that tissue •~he 16th day. Wound eontracture was not prevdnted, and the presc:nee of some dermis culture assay of frozen-,,ha,~ed skin is a deliwas,found tobe necessary to prevent these con- cate system which detects the selective dam'~ge tractures. This study shows that preservaiion o f epithelial, and fibroblast elements of the of epit,helial cells .alone cannot prevent graft skin. Both epithelial ceils and fibroblasts are shrinkage: I t is likely that, in the study of essential for optimal survival of skin as a Holst e,t s l Y ~hb cause Of shrinkage of rat graft.. Our aim was to find a procedure under skin 4 weeks artier grafting was, due to damage which frozen-thawed skin will grow out i n to the d e r m i s a n d fibroblasts as well as to; th6 tissue culture much like fresh skin, i.e., epiepithelial cells, This is confirmed by o u r in- thelial cells appearing? earlier than fibroblasts ability io culture either epithelial or fibroblast, and ,5oth types of cells appearing earlier than cells from'four of their human skin specimens day .6. Skin pretreaied in a medium containing 20 to. 30%: glycerol for 1 to 2 hrs at frozen and stored in the same manner. Perry e t al ~2.... Taylor , and. Gerstner] ~ 4°Cbefore freezing fulfilled this criterion. Although 0ur, initial observations were all Stenehe;~'er,:, HempeI, and Macintyre/'. McI(ee, Harris, a.!)d(I~ihan/~:,~and Allg0wer and Bl.ockm~ made on:hui:nan skin; the comparison betwc.,en ha~:'e utilized tissue culture methods to t e s t the tissue culture charac}erisfics and the behavior viability e[ ,skin S[ored in liquid nitrqgen,. The a s s gra)ft was performed (m rat skin in order authors.do n o t ;mept~i0n t h e type o f Cells or to get,. reliable :data on a larger scale than would, be possible .ruth human skin. Our earher their order. Of appearance. Both these methods; grafting and i¢issue cul- experiences / have shown' that 'the results of turei ~,:as/assays of :yi,~bility. ihave i/:advantages freezing r a t skin: ~have been applicable to huand: disadvantages: Grafting ,is: ~/simpler:: and manskin with very litt:!e modification. T h e tissue culture: characteristics of rat. skin more accurate, method of::evaluating the sur: TISSUE CULTURE CHARACTERISTICS OF RAT SKIN STORED IN DRY ICE AND IN LIQUID N ITROGEN .
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D I F F E R E N T I A L SUSCEPTIBILITY
frozen by two different methods show that the observations on human skin are valid. Skin frozen using the improved method (20% glycerol at --196°C) showed better preservation of epithelial cells than matching pieces stored using the older method (at --78°C in 10% glycerol). No shrinkage was noticed in skin treated with 10% glycerol a s expected from earlier~ results. Therefore~ we concluded that a freeze method-which permits outgrowth of epithelial cel!s in 50 to 60% spet:imens will probably provide viable skin for grafting. SU~IM:ARY
Fresh human sldn placed in plasma, clot culture shows outgrowth of epithelial cells within 3 days followed 3 to 6 days later by fibroblasts. :Plasma. clot culture of human skin pretreated in medium containing 10% glycerol at 4°C, frozen, and thawed showed outgrowth of fibreblasts only. Using this increased susceptibility of epithelial cells to freeze injury as a criterion, we developed a freezing method in which skin is pretreated with 20 to 30% glycerol at, 4°C for 1 to 2 hrs before freezing. Human skin preserved in liquid nitrogen by this method grew out in plasma, clot. culture very much like fresh skin. :REFERENCES 1. Allgower, M., and Blocker, T. G. Viability of skin in re]atioW to various methods of storage. Texas .Rep. Biol. Med., 10: 3-2I, 1952. 2. Ashley, C. A., and Bordley, J. The effect of freezing on the acceptance or rejection of autole~;ous rind homologous skin transplants. Transplant. Bull.~ 28: 27-30, 1961. 3. Baxter, It., and Entin, M. A. Experimental and clinical studies of reduced temperatures in in.iui'y and repair in man. III. Direct effect; of e0oling and freezing on various elemengs of human skin. Plast. Reeonstr. Surg., 3: 303-334, I948.
269
4. ]3erggren, R. B., and Lehr, H. B. Clinical use of viable;frozen human skin. J. A. M. A., 19~: 149-151, 1965. 5. Billingham, R. E . , and Medawar, P. B. The freezing, drying and storage of .mammalian ~ikin. J. Exp. Biol., 29: ,154-468, 1952. 6. Billingh~im, R. ~., and tleyn~lds, J. Tran~ plantation studies on sheets of pure epidermal epithelium and on epidermal cell susl~ension. Brit. J. Plast;. Surg., 8: 25-36, 1952. 7. Flatt, A. E. Ob~rvation:.,. on the growth of refrigerated skin grafts. Briu J. Plast. Surg., 3: 28-33.1951. S. Friedman-Kein, A. E:, Prose, P. H., Liebhaber, H., and Morrill, S. Culture of adult, human skin! In vitro grow~l and ker~,tinisation of epidermal cells. Nature, :~,.: ,o lo83--1584, -, , 1966. 9. Grille. t { . C., and McKhann, C. F. The ace~ptan(!e and evolution of dermal homografts freed of viable cells. Transplantation, 2: 4859, 1964. 10. I-Iols£. H., FeigI, P., Brown. A., and Lehr, l:I. B. Tempc:rature of additiveas a factor in eontracture of rat. skin autografts. Co'obiology. 2: 315. 1966. 11. MeKee, M. E., .Harris, S. E., and Kihara, It. Chromc~mM alterations with low temperature preservation of tissue fragmentsfor cultures. Prec. See. :Exp. Biol. Med., 123: 499502, 1966. 12. Perry. V. P , Evans, ~". J., Ityatt. G. W., Earle, W. 1:t.., and Drabeim, J. W. Transplant: Bull., 8: 50-52, 1956. 13. Pomerat. C. M. Pre~rvation and transplantation of normal tissues. I n C I B A Foundation symposium, G. E. W. Wolstenholme and M : P. Cameron, eds., pp. I7'.)--173. Little, Brm~m & Co., Boston, 1954. 14. Stenehever, M. A., Hempel, J. M., and Maeintyre, M: N . Maintenance o f iff vitro growth ability and chromosome integrity i'olIowing the deep freezing of minced fetal tissue. Cryobiology I : 240-241, 1965. 15. Taylor, A. C., and Gerstncr, R. Tissue survival after exposure to low temperatures'andthe effectivene~ of protect:ire pretreatment. I. Evaluation by gr0wth l'e, tis~.~m culture. J, Cell Comp. Physiol., ~6: 477-.502, 1955.
Our thanks to Mr. A. Brown and Mrs. Margaret Feret for their technieal assistance.
VOI. 5, No. 4, 1969
DIFFERENTIAL SUSCEPTIBILITY OF EPITHELIAL CELLS AND FIBROBLASTS OF HUMAN SKIN TO FREEZE INJURY R e p o r t o n an I m p r o v e d M e t h o d f o r S t o r a g e ot~ H u n m n S k i n at - 1 9 6 ° C ~
BALU It. A-THREYA, E. L. GRIMES, ttERNDON B. LEHR, ARTI=tUI-¢ E. GRE~ENE, AND LEWIS L. CORIELL
I.a~titute /or Medical Research, Camden, New Jersey, and HarH.~on Department el Surgical Research, University o/Pennsylvania School o] Medicine, Philadelphia, Pen'nsylvania years old. Skin was cut into ,approximately 1cm~square pieces. The pieces were placed in a medium in which they were to be frozen. At'ter 1 hr, ,the pieces were placed in sterile finger cots containing 3 ira1 of the corresponding medium. The finger, cots were then placed inside a sterile surgics,l glove and frozen in a Linde.BF-lfreezer at 1 to 2°C per man or in tl~e vapor phase of a liquid nitrogen tank at 7 to 8°C per mira When t h e temperature tions, stnnula,ted u s to study the effects of reached --100°C, the gloves were lowered into various •parsmeters of pretreatment and 5quid nitrogen and left there until ready for freezing/onyiabifity of epithelial and fibro- thawing. Thawing I was done. by agit'~ting the blastic elements of human skin by a "tissue finger cots rapidly ,in. water kept at 38°C. To test for viabiliW, eight to 10 small pieces culture' method. Prefimina:iT work indicated that, when (1 to 2 ram) of skin were placed in plasma fresh rat s k i n wasplaced in ~k plasma clot clot in plastic Petri dishes, covered with 5 ml ct~ture,"epithetial cellsgrew ou~ first, f'ollowed of Eagle's medium in Ea.rle's balanced salt 3 to :6: days later by a ring of fibroblasts at the solution with 10% inactivated newborn calf periphery.~However, when the same pieces were serum (EE medium), and maintained at 37°C treated,~vith" 10% glycerol a t 4 ° c for 1 hr, in a C0.~ incubator. Plates were read every fi.ozdn~(:ith~Wed,:and cultured, fibroblast~s grew 3rd day or more frequently, and the nature of out at about:.the same ,txme but ep~tnelml outgrowth was noted. The outgrowth was graded from 1+ to 4+. When the edge of a ceils we?e:del~e4 or absent. Tn/thiS rel~o~t:}'evidence is presented to show piece of skin was focused under 100X of an their: freez~ng"of:~fUll thickness adult human inverted: microscope~ witli the edge of skin stdn at. ~1967C ::results :in :more damage to visible in one~6orner, the presence of only ep~tSeliaLeellS::than.:tOtheifibroblasts and that seven to eight cells or fewer was rated as #itheiial ~eJemeli~ of :skin. are better preserved "occasional outgrowth/' When the skin ~howed iii: Skiii t)retreated 'with a medium containing outgrowth of cells reaching 25% of the .area visible under the microscope, it was graded 1+. 20' i030% glycer01 for ~It0 2hrs at 4°c. When the cells covered .~A)%of the area, it was 2 + ; i f the cells covered 75% of the area, it !-~V~a.TERIAL$ AND ~ E T H O D S was 3 + ; and if the cells covered the entire Skin was obtained from patients :undergoing elective su~geo-.:M6s~: of. tile pieces w'ere :from area visible, it Wa~s4+. the ~ abdomen"of patieiits ~i~)et(ve6n30 and 40 RESULTS
~ o l ~ et al.'° observed that rat skin soaked in 10% glycerol or 10% dimethyl sulfoxide (DMSO) for 2 hrs at 24°C and cooled to 4°C before, freezing decreased i n size by about 25% When, grafted back to the same animal, whereas nonfrozen controls showed no shrinkage~ when huma~ skin stored at ,-196°C in a medium Containing 10% glycerol was placed on piasma :clot culture, there was no outgrowth of t
:Before ~freezing, explants of human skin Receiyed August ,14i 1968. * Tl~is:'inves~igati~h:,WaS;Supportedin par~ bY a showed/an initial outgrowth of epithelial cells gran~ (fr~m,the 30hn~A.-'Harfford.Fo~mdatSon and as early as 3 days in culture, and fibroblasts in par t b y P u b l i c Health Serkice Research Gran~ grew out by about the 6th day. The final CA-0~!953--09 from the ~Na~mnal " " ' ' Cancer Institute. 262
DIFFERENTIAL SUSCEPTIBILITY SKIN VI~'~iLITY
203
STUDIES EPffHELJAL- UNFROZEN 4-t EPITHELIAL-FROZEN "RBROBLA~- UNFR(IZEN RBROBLAST- FRffZEN
:c
4
f
o iI:
(D 2÷
-=:-::~
0
f
IH
3
6
9
12
15
18
DAYS AFTER CULTURE
Fio. 1. Comparison of tissue culture characteristics of fresh human skin with those of a matching piece frozen after pretrcatment with 10% glycerol at 4°C for 1 hr. Note that in unfrozen skin epithelial cells ap!sear very early and make a full sheet whereas fibroblasts appear later. In matching pieces of frozen-thawed skin fibroblasts appear iust as in unfrozen skin s~ad make a better sheet than in unfrozen skin. Epithelial cells appear after 12 days and make a smaller sheet. (Grades 1 to 4 are described in text.) outgrowth of epithehal cells was usually 4+, and that of fibroblast, usually 2+. However, when human skin was treated with 10% glycerol or 10¢o DMSO for 1 to 4 hr,s at 4°C and then frozen, thaived, and cultured, the epithelial cells did not come out until about t0 to 12 days after culture. Fibrbblasts started growing by the 6th day of culture similar to unfrozen sldn,-and the final outgrowth of fibroblasts was generally 3+ (Figs. 1, 2, and
3). Duration o] pretreatment. Skin was cut into 28 pieces and left in medium containing either 10% glycerol or 10% DMSO for V2, 1, 2, 4, 8, and 24 hrs at 4°C before freezing. Skin cultures were prepared before and after freezing. Pieces of skin pretreated in glycerol or DMSO for less than 1 hr before freezing showed outgrowth of cells after 12 days i n culture. When the skin w a s pretreated with 10% glycerol or 10% DMSO for 1 to 4 hrs before freezing, there was outgrowth of cells within 6 days. Skin. left in glycerol or DMSO for 8 and 24 hrs before freezing grew out in 8 days. No difference could be demonstrated between the crYoprotective effects of 10% glycerol and 10% DMSO by these techniques. Temperature of pretreatment. Pretrea/mcnt of the skin with 10% glycerol for 1 hr at
4~C was compared with pretreatment at 370C to determine whether glycerol might more easily penetrate the layers of the skin at 37°C and hence protect the epithelial cells. The results sununarized in Table 1 show t h a t there was no better, survival of cells exposed to glycerol at 37°C.
Rage of /reezing on viability of epithelial cells. The effect of rate of freezing on viability was tested as follows. A piece of skin was cut into two halves. One half was frozen i n 10% glycerol at the rate of 7 to 8°C per rain. The other half was frozen rapidly at the rate of 125°C per min. When thawed and cultured; both pieces showed similar outgrowth of cells and .the fibroblasts appeared 3 to 4 days before tile epithelial cells as i n earlier experiments. tIyaluronidase ]or pretreatment. To ,facilitate the- penetration of t h e tissues by : glycerol, hyalur0nidase was , added to m e d i u m containing 10% glycerol. The skin biopsies were ex~ posed ,t° these, m e d i a Containing 10 unitS:,per ml and 50 traits per ml of hyaluronidase at 4°C for 1 hr, before freezing'. Under o~r,:ex,perimental condit, ion; ~he delay ,!n onset ',of growth ,,of epithelial cells: Still,-per~isted, 'irre,spective of t h e presence or absence of hyaluronidase.
264:
ATHREYA ET A L.
l,'~a. 2. Epithelial cell sheet from plasma clot culture of unfrozen human skin.
Varying concentrations of glycerol and epithelial viability. Varying concentrations of glycerol were tested by immersing pieces of skin in E E medium cont.qining 0 to 50% glycerol for 1 hr at 4°C. A small portion of each of these pieces was used for immediate culture. The remaining portion was frozen at the rate of 7 to 8°C per rain. When thawed and cultured, skin frozen in 20 to 30% glycerol showed the most consistent viability (Figs. 4, 5, and 6). Epithelial cells consistently grew out earlier than fibroblasts and formed a riffler sheet after pretreatment with 20 and 30% glycerol m~d eve~ with 40 and 50% glycerol, occasionally. ~'nese experiments were repeated with 20% glycerol at 4°C to detemdne the optimal treatment time before freezing. The results shown in Table 2 indicate the optimal time to be between 1 and 2 hrs as shown by better survival of epithelial cells.
Transplantation of frozen skin. In order to compare the ability of frozen-thawed skin processed by this improved method to grow in tissue culture with its ability to function as viable graft, the following experiment was done. A piece of skin 2 inches by 1 inch was removed from the ventral surface of 15250-g Amsterdam rats and cut into two halves. One half was treated with E E meclium containing 10% glycerol for 2 hrs at 4°C and frozen in glass tubes in 5 mI of the same medium in a Dry Ice chea at --70°C. The other half was immersedin E E medium containing 20% glycerol for 1 hr at 4°C and frozen in the vapor phase of liquid nitrogen. Both pieces were thawed after 24 hrs by agitating in water at 37°C and transplanted to the dorsum of the original donor rat. The grafts were measured on day 0 and on day 28. Plnsma clot cultures of both halves of skin were done before and
DIFFERENTIAL SUSCEPTIBILITY
,.t)o
Fro. 3. Plasma clot culture of human skin (same as in :Fig. 2) but after freezing and thawing. Prctreatment with 10% glycerol at 4°C for 1 hr. Note only fibroblasts. after freezing and were analyzed for outgrowth of epithelial cells and fibroblasts, the day of appearance, and the extent of growth. In all the cultures of sldn before freezing, epigleIitfl cells grew earlier than fibroblasts and made full sheets earlier. This pattern was seen also in skin frozen in 20% glycerol at -196°C. Ilowever, matching pieces of skin frozen in 10./o glycerol at - 7 0 ° C showed early outgrowth of epithelial cells in 50% of the cases, tile fibroblasts growing out earlier in tl~e others (Fig. 7). All of the grafts took well, and we could not confirm the earlier finding of graft shrinkage. f, t
TABLE 1 ~ F F E C ' r OF ])RE'rltEATMENT OF SKIN IN ~IEOIU~t
4°C .,,','u ,~'r 17°C (POS'rm~EZE CVLTm~ES)
COXT.,,IxlXG 10% GW,'CnROL
~. pedment No.
of"
Onset of
0dsct
oi Fibreblasts
o£ Epithellal Cel!
Final Type
E:~tent of Growth
F 1,2. F
4E, 4F
I
1
4 87
2
4 B7
3
-i B7
I)lsc~'ssIox Assay of viability of skin after any storage procedure is ~ difficult problem. Lerggren and Lehr,' Flatt7 ]3illingham and Medawar; ~ Ash-
Te.mperature
.vc
14 11
6
6
14 11
17
I E, F i E, F i E, F 10 E, F t
* For explanation of s:~m~bols see text.
l+*
4E, 4F 4E, .IF 4E, F4 4E: 4F
A
EPITHELIAL CELLB FIBROBLASTS
40
50
m=.
o,
tO 0 :%:
0 ~o
o 0 ,0 ~o t5
I0
20
30
PERCENTAGE OF GLYCEROL IN THE MEDIUM
Fro. 4. Effects .of 'various concentrations of glycerol in the, pretreatment medium o n the viability of epithehal, cells and fibroblasts (summal'y of 1Oexperiments). Note that, until the glycerol concentration is increased to 20%, fibroblasts always appear earlier than epithelial cells. (In unfr0zen skin, epithelial cells alwuys appear earlier.) FREO,U]@IIC~/"OF ONSI~I' OF OUTGROWTHSOF FIB~OILLASTS ~ CELLS ~
A~'~'D~,P.ITRELI.AL
BFJ~RE DA'I 6 FROM ~I~OZEN-TRAW~ HURAN SKIN
g= o
o3
!.4 0 ,.--1
0 0
I0
2O
3O
4O
5O
P~CE~IT OF C~LI~OL IN THE Y2~IUM
Fm. 5~ Effects of-yangus:concentrations of glycerol in t h e pretreatment medium on the viabi!i~.Y:of,: epithelial: ce!Is !and~ fibrobl~ts: .In order to obtain, condition :.under which epithelial ::cells Can:appear earlier than -6 (lays in tisSue Cultare,: t.lii; glycerol concentration has to goOver20%. 266
I)IFI, EREN TIAL St,,SCLI TIBILI r~2
2G7
~ORRELATIOI~ BETWEEN PERGE2CFA~EOF GLYCEROL IN THE PRETRF.ATHENT&RI} FREEZE NEDIIJH AND SIJCCESSFUL RI~OV~t¥ OF FIBROBLA~TS Ah'D EPITHE1/IAL CELL~ FROH FROZEN-THA~ HUHAN SKIR FIBROI~L~TS
!
1
o
re:
g
(9 a0
o U
¢.9
Cq
10
2.0
3O
50
PF/ICEI~',.~GE OF GLYCI~IOL IN THE N~.~IUH
FI¢;. 6. Effects of various concen.tm~_ons of.glycerol in the pretreat,ment medium on the successful outgrowth of epithelial cells and fibrob!astS'on or about~ the 18th day of culture, In this graph, every piece of skin which showed an outgrowth of epithelial cells or fibroblasts was graded as positive, irrespective of the number of days it took for them to .first appear in culture. (In cultures of Unfrozen skin, there is ofltgrowth of epithelial cells and fibroblasts in every instance.) After fl-eezing and thawing the optimal recovery" of both types of cells is 90% when pretreated with a medium containing 20 to 30% glycerol. qhkBl.,l~ 2 I~FFEC'I'S OF ]~ I~L [I,l:,Aq M]~IN 1. OF ]I:IUMAN ~ K I N W I T H
[20%'GLYCEROL a'r-4°C rOit \:AllYINGDURATIONS OF T I M g BI,3FORI~. F . R E E Z I N G
Duration of Pre trea tmen t
Ceil T ~ e to Appear Early in Tissue Culture ~
No, o{ Experiment
Epithelial {Fibroblasts
%
(. 's)
5
0 40
5
40
5 2 2
20
5
1 2 4 8 24
r
100 80 100 ,
100
0
0
80
,
100
* In culfures of .unfrozen skin epithelial cells appear first, in every instance, ley and.Bordley,~ and. Baxter and, Entin~have used .the microscopic appearance of t h e graft for establishing criteria for survival. Billingiaam: a n d Medawar~ established the
following criteri~ for evidence of survival of skin grafts: " ( a ) ~kpI)earanCe of an. mmult~s-:of migratory outgrowth f r o I n t h e , origina! graft, margin over ithe surrounding raw area.... (b) SUr~dval of epidermal melanoblasts..(c)..Preservation of fine, fibrous. orgamsatlon., " ' """ " Using these criteria, -they found-slow freezing foll0wedby rapid thawing was best: for rabbit skin and found glycerol concentrations between"I0-a,nd 3 0 % asl being effective- in reducing ,freeze;ir/2 jury. They i'otmd that whole skin.su'rvivedex: p o s u r e . a t 0°C to" 98.t% ,glycerol-for.. ' -" :8 -lira a n d to 8 0 % .gtyeero! f o r 2 hrs; whereas: a suspension of epithelial cells showed damage.. ag these concentrations of glYeeroI after 35 .ram. In oar studies;...Skin Soak(id :'in:.lOO%:glyeero1 for :1 h r : at.. 4? C. felt" like .:a..piece,"iof CardbOard bu~"sti!Fshowe d ..outgrOwth.. of ePithetiai cells and fibroblasts,bef0re ~,md afterfreeZing. Bminghm :and"J,eynotde: fised i-:pure. :, epidermal 'sheets ~and suSpensi0n'.:of.iepidielihi' ceiis to cover., areas :of skin,.Ioss':in rabN,ts:":The',; '
.
.?
268
ATtIREYA ET AL.
vivM of skin. It will test not only for cell survival but also for the ability of skin to function as an organized tissue. Skin need not be viable to be effective as a i00 temporary graft and, therefore, freeze-thaw studies of human skin evahmted solely on the gross appearance are not a final criterion of o 75 viability as discussed by Grfilo and h , Khann. ' In our own studies we have shown that four pieces cf human skin from the skin bank 5o of the Hospital of the University of Pennsylo vania showed no outgrowth of cells in tissue culture, although they had functioned ade25 quately as grafts. Tissue culture assay of frozen skin is a C~ simple in vitro assay. :Tile conditions under which the cells have to grow are less favorable F 18RO BL AS TS EPITHELIAL but perhaps more reproducible than when Early Outgrowth grafted onto an animal. In tissue culture, of Cell Types Before Day 3 identification of cells as epithelial cells or Fro. 7. Note that, when stored in 20% glycerol fibroblasts is usually easy. Using electron microscopic study of skTn in plasma, clot culat --196':C, epithelial cells grow o u t e a r l i e r than fibrobtasts, and before day 3 in 90% of samples. tures Friedmar~-t.(ien et al.'~ concluded that, in This is similar to the unfrozen skin. Epithelial the immediate vicinity of the explant, outcells appear earlier in only 50% of samples frozen at --70°C ,xfter pretreat.menl; in a medium con- growfll of epithelioid cells does not necessarily mean absence of fibrob]asts. A]so, in ti~ae taining I0% glycerol. culture of skin a cut sweat gland duct-,may gave good, temporary protection, but the grafts proliferate and show a good-outgrowth of separated from the underlying tissue by about epithelial cells and give false resultsY The results of this study shm~ that tissue •~he 16th day. Wound eontracture was not prevdnted, and the presc:nee of some dermis culture assay of frozen-,,ha,~ed skin is a deliwas,found tobe necessary to prevent these con- cate system which detects the selective dam'~ge tractures. This study shows that preservaiion o f epithelial, and fibroblast elements of the of epit,helial cells .alone cannot prevent graft skin. Both epithelial ceils and fibroblasts are shrinkage: I t is likely that, in the study of essential for optimal survival of skin as a Holst e,t s l Y ~hb cause Of shrinkage of rat graft.. Our aim was to find a procedure under skin 4 weeks artier grafting was, due to damage which frozen-thawed skin will grow out i n to the d e r m i s a n d fibroblasts as well as to; th6 tissue culture much like fresh skin, i.e., epiepithelial cells, This is confirmed by o u r in- thelial cells appearing? earlier than fibroblasts ability io culture either epithelial or fibroblast, and ,5oth types of cells appearing earlier than cells from'four of their human skin specimens day .6. Skin pretreaied in a medium containing 20 to. 30%: glycerol for 1 to 2 hrs at frozen and stored in the same manner. Perry e t al ~2.... Taylor , and. Gerstner] ~ 4°Cbefore freezing fulfilled this criterion. Although 0ur, initial observations were all Stenehe;~'er,:, HempeI, and Macintyre/'. McI(ee, Harris, a.!)d(I~ihan/~:,~and Allg0wer and Bl.ockm~ made on:hui:nan skin; the comparison betwc.,en ha~:'e utilized tissue culture methods to t e s t the tissue culture charac}erisfics and the behavior viability e[ ,skin S[ored in liquid nitrqgen,. The a s s gra)ft was performed (m rat skin in order authors.do n o t ;mept~i0n t h e type o f Cells or to get,. reliable :data on a larger scale than would, be possible .ruth human skin. Our earher their order. Of appearance. Both these methods; grafting and i¢issue cul- experiences / have shown' that 'the results of turei ~,:as/assays of :yi,~bility. ihave i/:advantages freezing r a t skin: ~have been applicable to huand: disadvantages: Grafting ,is: ~/simpler:: and manskin with very litt:!e modification. T h e tissue culture: characteristics of rat. skin more accurate, method of::evaluating the sur: TISSUE CULTURE CHARACTERISTICS OF RAT SKIN STORED IN DRY ICE AND IN LIQUID N ITROGEN .
"
'
. r ' ) '
.
'
'"
,"
,
"
'*'
,':
'
':
'
"
'
"
.
.
.
.
'
.
.
'
.
"
_
"
"
"
D I F F E R E N T I A L SUSCEPTIBILITY
frozen by two different methods show that the observations on human skin are valid. Skin frozen using the improved method (20% glycerol at --196°C) showed better preservation of epithelial cells than matching pieces stored using the older method (at --78°C in 10% glycerol). No shrinkage was noticed in skin treated with 10% glycerol a s expected from earlier~ results. Therefore~ we concluded that a freeze method-which permits outgrowth of epithelial cel!s in 50 to 60% spet:imens will probably provide viable skin for grafting. SU~IM:ARY
Fresh human sldn placed in plasma, clot culture shows outgrowth of epithelial cells within 3 days followed 3 to 6 days later by fibroblasts. :Plasma. clot culture of human skin pretreated in medium containing 10% glycerol at 4°C, frozen, and thawed showed outgrowth of fibreblasts only. Using this increased susceptibility of epithelial cells to freeze injury as a criterion, we developed a freezing method in which skin is pretreated with 20 to 30% glycerol at, 4°C for 1 to 2 hrs before freezing. Human skin preserved in liquid nitrogen by this method grew out in plasma, clot. culture very much like fresh skin. :REFERENCES 1. Allgower, M., and Blocker, T. G. Viability of skin in re]atioW to various methods of storage. Texas .Rep. Biol. Med., 10: 3-2I, 1952. 2. Ashley, C. A., and Bordley, J. The effect of freezing on the acceptance or rejection of autole~;ous rind homologous skin transplants. Transplant. Bull.~ 28: 27-30, 1961. 3. Baxter, It., and Entin, M. A. Experimental and clinical studies of reduced temperatures in in.iui'y and repair in man. III. Direct effect; of e0oling and freezing on various elemengs of human skin. Plast. Reeonstr. Surg., 3: 303-334, I948.
269
4. ]3erggren, R. B., and Lehr, H. B. Clinical use of viable;frozen human skin. J. A. M. A., 19~: 149-151, 1965. 5. Billingham, R. E . , and Medawar, P. B. The freezing, drying and storage of .mammalian ~ikin. J. Exp. Biol., 29: ,154-468, 1952. 6. Billingh~im, R. ~., and tleyn~lds, J. Tran~ plantation studies on sheets of pure epidermal epithelium and on epidermal cell susl~ension. Brit. J. Plast;. Surg., 8: 25-36, 1952. 7. Flatt, A. E. Ob~rvation:.,. on the growth of refrigerated skin grafts. Briu J. Plast. Surg., 3: 28-33.1951. S. Friedman-Kein, A. E:, Prose, P. H., Liebhaber, H., and Morrill, S. Culture of adult, human skin! In vitro grow~l and ker~,tinisation of epidermal cells. Nature, :~,.: ,o lo83--1584, -, , 1966. 9. Grille. t { . C., and McKhann, C. F. The ace~ptan(!e and evolution of dermal homografts freed of viable cells. Transplantation, 2: 4859, 1964. 10. I-Iols£. H., FeigI, P., Brown. A., and Lehr, l:I. B. Tempc:rature of additiveas a factor in eontracture of rat. skin autografts. Co'obiology. 2: 315. 1966. 11. MeKee, M. E., .Harris, S. E., and Kihara, It. Chromc~mM alterations with low temperature preservation of tissue fragmentsfor cultures. Prec. See. :Exp. Biol. Med., 123: 499502, 1966. 12. Perry. V. P , Evans, ~". J., Ityatt. G. W., Earle, W. 1:t.., and Drabeim, J. W. Transplant: Bull., 8: 50-52, 1956. 13. Pomerat. C. M. Pre~rvation and transplantation of normal tissues. I n C I B A Foundation symposium, G. E. W. Wolstenholme and M : P. Cameron, eds., pp. I7'.)--173. Little, Brm~m & Co., Boston, 1954. 14. Stenehever, M. A., Hempel, J. M., and Maeintyre, M: N . Maintenance o f iff vitro growth ability and chromosome integrity i'olIowing the deep freezing of minced fetal tissue. Cryobiology I : 240-241, 1965. 15. Taylor, A. C., and Gerstncr, R. Tissue survival after exposure to low temperatures'andthe effectivene~ of protect:ire pretreatment. I. Evaluation by gr0wth l'e, tis~.~m culture. J, Cell Comp. Physiol., ~6: 477-.502, 1955.
Our thanks to Mr. A. Brown and Mrs. Margaret Feret for their technieal assistance.