Biologic Crystals and Particles Produced in Tissue Culture*

I . Introduction

GEORGE G. ROSE

(Departmeni of Biology, Univernty of Texas M. D. Anderson Hospital and Tumor Inifitute; Deparimen@ of Medicine, Universityof Texas Dental Branch; and Tissue CuUure La&ratory, Herinann Hospital, Houston, Texas)

SUMMARY

A method for the cultivation of embryo chick tissues which gave rise to numerousand unusually large crystals and particles is detailed. Forms were helical, tubular,ribbon-like, hexagonal, rhomboidal, and filamentous. Rhomboids and hexagonals oftenhad surface areas several times the size of the broad and flat tissue-cultured cells, andhelices were observed up to 300@ in length. Transformations of helical and relatedforms were observed. Their chemical identity could not be determined by conventionalcytochemical technics. With modifications, however, helical and related forms wereshown to give a positive reaction to pyronin staining which was inhibited by priordigestion in ribonuclease.

In the summer of 1959, multipurpose culturechambers (14, 15, 24) were fabricated with confronting cultures of 11-day embryo chick thyroidand lung tissues. These explants were completelyseparated from the nutrient vault of the chambersby interposing sheets of dialysis cellophane (19—21, 23) and thereby differed from cultures established by the formerly described cellophane striptechnic (24). The strip technic permitted a flow ofthe nutrient around the edges of the cellophane(2, 22, 25), so that the explantswerepotentiallyaffected by all the ingredients in the nutrient,whereas the sheet technic permitted only a dialysate of the nutrient into the culture environment.

After a month of cultivation the thyroid andlung tissues appeared to have reached a staticstate with respect to their degree of emigrationand population mass. Also, cells in the originalexplant sites were highly differentiated. The explants still appeared to be unaltered at 3 months,although their contours were gradually changing.Smooth muscle activity in the lung was viewed as

* T@ work was supportedin part by Research Grant(CA05100-03CB) fromthe National CancerInstituteof theNational Institutes of Health, U.S. Public Health Service;grant-in-aid GB-is continuation of G-1409i from the NationalScience Foundation administered by C. M. Pomerat of thePasadena Foundation for Medical Research; and grant-in-aidfrom the Easter Seal Research Foundation, National Society

for Crippled Children and Adults, Inc.

Received for publication September 4, 1965.

a “lazy―nonrhythmic contraction which perforceaccounted for the contour change. Rhomboidaland filamentous crystalline forms also were notedaround the lung explants, and the epithelial arrangements in both the lung and thyroid remainedhighly differentiated.

At 6 months of cultivation the explants againwere relatively unchanged, except for contour, andsmooth muscle activity persisted. It was then decided to retain these cultures to determine howlong differentiation would persist before their demise or transformation to a neoplastic growth.Other workers (12, 26) had indicated that tissuecultures, regardless of their origin, reverted toneoplastic strains if kept sufficiently long; yet itappeared that these embryo chick cultures isolatedby the cellophane molecular screen might becomean exception to this usual conclusion. Smooth muscle contractions persisted for 10 months of cultivation.

Finally, the explants were retained for 25 monthswithout histologic evidence of neoplastic growthor changes in the total cell masses. Although thefinal tissue organization was compact and withoutthe well defined architecture observed in the youngcultures, the emigrating cells were of a “normal―character, and mitoses were not observed.

This and subsequent experiments which utilizedembryo chick tissues separated from the nutrientvault by sheets of dialysis cellophane revealed sev

279

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280 Cancer Research Vol. 23, February 1963

en major points of interest : (a) cultures did notshow histologic evidence of neoplastic growth; (b)mitosis was markedly reduced ; (c) normal function, when evident, was prolonged; (d) degenerative phenomena were minimized ; (e) fibroblasticout,growths were diminished; (f) fat droplets in thefibroblasts were significantly reduced or eliminated; and (g) cultures from many types of tissuesultimately contained crystalline and .particulatearrays of varying sizes and shapes. Three generaland provisional groupings of these megaparticleshave been categorized according to their origin,morphology, and behavior : (a) tubules, helices,and ribbons; (b) rhomboids and hexagonals; and(c)filamentousforms.

This preliminary report details general descriptions of the form and size of the observed megapartides by phase-contrast and bright-field (hematoxylin-stained) micrographs. Time-lapse sequencesalso are used to denote : (a) relationships of cellsto crystals; (b) malleability of helical forms; and(c) transformations of the particles occurring overan interval of time. Responses to cytochemical,solubility, and thermal tests are presented.

MATERIALS AND METHODS

Specimens.—Embryo chicks incubated for 11—19 days were used to procure explant material forthese experiments. Specimens were dissected fromthe cerebrum, cerebellum, spinal cord, thyroid,lung, pancreas, gonad, kidney, proventriculus,large intestine, small intestine, striated muscle,and leg bone (minus periosteum and endosteum)areas.

Cultivation procedures.—Standard tissue cultureprocedures were used to open fertile eggs, removeembryos, and dissect specific organs and tissues.Explant specimens were cut into pieces approximately 1 mm. X 1 mm. with sharp scalpels inPetri dishes and washed with Fischer's V-614 fluidnutrient minus the serum component. Four to sixexplants were placed on coverslips of 2 X 3-inchmultipurpose culture chambers (14, 15,24) and coyered with sterile pieces of cellophane (19, 20, 23)moistened with Earle's balanced salt solution(BSS). The cellophane completely enclosed the tinsue fragments, so that when the rubber gaskets andclosing coverslips were placed in proper positionthe completed chamber was divided by the cellophane into two areas : (a) a culture vault and (b) anutrient vault. In this way serum constituents ofthe fluid nutrient which were not dialyzable didnot come in contact with the explants, and cellularproducts of large size similarly did not leave theculture environment through the cellophane pores,their diameter being approximately 24 A. Some

chambers were established without tissue fragments, and other chambers were established withtissue fragments and with four No. 25 hypodermicneedle performations in the cellophane. This lattercondition produced a chamber construction inwhich (a) all serum constituents of the nutrientcould pass to the explant environment and (b) allcellular products could pass to the fluid nutrientvault and then out of the chamber when nutrientswere exchanged.

Nutrient.—The fluid nutrient utilized in theseexperiments was a standard type of tissue culturepreparation (19—21, 23) composed of Fischer'sV-614 (75 per cent) ; calf serum (20 per cent) ; andwhole egg ultrafiltrate (5 per cent). Chambers werefilled with this nutrient immediately after fabrication and placed in a 37°C. incubator. Fluids of thenutrient vault were completely exchanged semiweekly by syringe technics through the rubbergaskets of the chambers. Culture vaults were notdisturbed and were affected, therefore, only bymolecules small enough to pass through the dialysin cellophane screen.

Observaiions and photography.—Cultures wereobserved with Bausch and Lomb phase@ontrastmici@oscopes and recorded by both still photography, with a Hasseiblad 2@X 2@camera, and timelapse cinemicrography, with a 16-mm. EMDECOtime-lapse unit (13, 16—18,2.5).

MICROCHEMISTRY

Cytochemistry.—The nutrient of cultures withmegaparticles was evacuated and replaced withBSS. After this rinsing, the BSS was replaced byformol for 20 minutes. This was followed by a29-minute water rinsing. Staining procedures werecarried out through the cellophane by admittingreagents into the nutrient vault with the syringeneedle which pierced the rubber gaskets, and byextending time intervals to permit adequate penetration through the cellophane screens. Positivecellular reactions were used to certify a satisfactory procedure for the following tests : generalstaining with acid hematoxylin (Delafield's), Feulgen's DNA (8), Brachet's RNA-DNA (1); basicprotein dyeing with toluidine blue (pH 8—9)andunmordanted hematoxylin (pH 8.2) according toFullmer and Lillie (4) ; polysaccharides with thePAS method of I{otchkiss (10) ; neutral fats withSudan IV in carbitol according to Gomori (5);sterols-steroids with the Schultz-Smith test forcholesterol and its esters (8) ; and acid phosphataseaccording to Gomori (6). Vital dyeing using Janusgreen, neutral red, and acridine orange was effectedalso. Stronger concentrations of vital dyes thannormally used were necessary to hasten the pene

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ROSE—Biologic Particks in Tissue Culture 9281

tration of the cellophane. Here again, cellular dyeing certified a satisfactory penetration of the cellophane by the dye; hence, its availability to themegaparticles.

In addition to the conventional cytochemicaltechnics enumerated above, nutrients were removed and replaced by a 92per cent aqueous solution of KMnO4 for 1 hour. This fixed and lightlystained the crystalline and particulate forms but,more importantly, permitted a separation of thecellophane from the coverslip without severelydistorting the tubules and helices. Formalinfixed tubules and helices were destroyed or floatedaway when the cellophane was peeled off the coyerslip. Potassium permanganate fixation resultedin an attachment of the particles to either thecoverslip or the cellophane. Tubules and helicesattached to the coverslips were subjected to pyronin staining for RNA according to the methodofBrachet(1).

Solubility.—Nutrients were washed out and thesolvents injected into the nutrient vault. In allcases the solvents came into contact with the partides by passing through the cellophane screen.Cellular response certified presence of the solventaround the megaparticles.@ The following solventswere used at room temperature : methanol (100per cent), ethanol (50 per cent and 95 per cent),chloroform vapors, distilled water, HC1 (M/1),NaOH (Mu), and NaC1 (M/1).

Thermal effects.—Fluid nutrients were replacedwith BSS and a vent needle left in the nutrientvault. Chambers were then immersed in a waterbath at 50°C., and then at increments of 10°C.for 20-minute periods.

RESULTSD@scRwrIvE MORPHOLOGY

A general orientation and comparative analysisof crystalline and cellular size may be obtainedfrom the low-power micrographs in Figures 1through 4. These show outgrowths from 11-dayembryo chick cerebrum after 58 days of cultivation and their association with sperific types ofcrystalline accumulations. Tubules, spirals, andribbons are shown in Figure 1 ; rhomboids in Figure 92; small and refractile hexagonals in Figure 3;and filamentous forms in Figure 4. Crystals appeared as early as 8 days after cultivation hadcommenced, though more commonly several weekselapsed before many became apparent. All the tissues investigated, with the exception of leg bone,produced crystalline displays to varying degreesas shown in Figures 1 through 58.

Tubules are shown at low and high powers inFigures .5 through 9. The emigrating fibroblast (fb)

of a 19-day embryo chick gonad (Fig. 5) isshown at low power after 27 days of cultivationsurrounded by a sea of needle-like tubules. Generally, the tubules rested on the surface of thecellophane ; but, when the coverslip and cellophanewere closely apposed, crystals and cells were in thesame plane so that contacts were unavoidable.Tubules often became entangled in debris and appeared in clusters as those shown at high power(Fig. 6) from a 35-day culture of a 12-day embryochick stomach. Note their uniform diameter. Macrophages and other wandering cells frequentlybecame closely associated with tubules. The cellin Figure 7 sent probing extensions along a tube,both to the left and to the right, as it passed overit. Small granular masses sometimes were observedalong tubes of old cultures (Fig. 8) and occasionally, sharp angular bends or angular attachmentsbetween tubules were noted (Figs. 9, 28).

Spirals of Figure 1 are shown again at a mediumpower in Figure 10 after 58 days and again inFigure 11 after 76 days of cultivation. The cellophane scratches along the right margin of thesemicrographs made convenient markers for thetransformations which occurred. The large centralspiral A became longer and its ribbon width broader; spiral B also elongated. The four finger-likeprocesses of ribbon C similarly elongated and theribbon to the far right with a few spirals whichcrossed the cellophane scratches in Figure 10 gently unfolded during this interval (Fig. 11). Otherhelical elements from the same cerebral culture areshown after 58 days in Figure 12. Spiral D had abroad unwinding in its central portion, and spiralE appeared to have nearly completed an unwinding. Other stages of spiraling are shown though notspecifically labeled.

Helical forms were lightly stained with Delafield's hematoxylin after formol fixation and a prolonged (24 hours) staining in the chamber throughthe cellophane. The micrographs of hematoxylinstained helices in Figures 13 through 16 derivedfrom 11-day embryo chick gut after 62 days ofcultivation are shown in unusual arrangementssuggesting duplication processes. Pairs of spiralswere noted to coincide precisely, and in Figure 16there are three coinciding spirals. In Figure 14 theribbon of the major spiral F appeared denser in itsleft extremity than its right, suggesting that thelower portion of the pair (G) peeled from the upperor major portion F.

Other suggestions of replication are shown in thephase-contrast micrographs in Figures 17 through19. A pair of spirals or a single spiral which hadsplit down the middle of the ribbon is shown inFigure 17. A spiral which had split at one end and

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Cancer Research Vol. 923, February 196392892

unwound as two lesser-sized spirals is shown inFigure 18. The tubule in Figure 19 appeared tohave unwound in its upper extremity, and a secondspiral to the left coincided with it in the same spacing arrangement as those of Figures 13 through 16.

Figures 21 and 23 are micrographs of two tubules at a focus through their mid-portion. It wasobserved that these tubules were not smoothwalled but were periodically dashed with dark areasin alternate positions on opposite sides of the tubules. In reality periodic dashes were thickeningswhich coincided with the pitch of the early-formingspirals observed at a high focus (Figs. 20, 22). Thetubule of Figures 22 and 23, which is the particleshown in the sequence of Figures 34 through 38,had commenced to unwind at both ends.

Generally, the tubes and spirals were observed asstraight forms, although occasionally they werebent or bowed by migrating cells or even by agentle finger pressure on the coverslip. The loosespiral in Figure 24 had formed a loop and remainedunchanged during a week of observation. Thespiral in Figures 30 and 31 shows the straight andcurved position induced by finger pressure on thecoverslip. Tubules snapped back to the straightposition rapidly after being bent by finger pressure,and spirals returned more slowly, if at all.

Other unusual forms of the tubes and helices areshown in Figures 2.5 through 29, and in Figures 32and 33. In the series, Figures 2.5through 27, a helixis shown enveloping a tubule. This form was oh

served with some regularity, though it was a sparsefinding among the many other particles. The micrographs were made at three focal levels to showunequivocally that the helix enclosed the tubule.The high focus in Figure 2.5 denotes the righthanded spiraling which was characteristic of allembryo chick helices observed. The long tube Hin Figure 928had a sharp bend at the top, globularinternal images in the center and at one side, anda spiral unwinding of the lower extremity. In thissame figure, two other tubules (I and J) are shownin a partially unwound state. The large tubule Kin Figure 29 had partially unwound to a greaterdiameter at its lower portion than generally ohserved. In the lower right, tubule L is shown withspiraling surface images as the tubules in Figures20 and 22. One of the more unusual relationshipsbetween tubules and spirals is shown in Figures32 and 33. In this instance, one tubule (M) penetrated another tubule (N). The upper half of Nhad uncoiled, producing a situation comparable tothat in Figures 2.5 through 27. The image of apiece of a very wide helical form focused at themid-region@is labeled 0, and a high-power view of

the helical portion of N surrounding the tubule Mis shown in Figure 33.

A relationship between particulate or crystalline formation and cellular activity was not ohserved. However, on many occasions, particulateand crystalline elements were observed in closeproximity with cells which often disturbed theirgeometrical form by breaking tubes or unwindingspirals. One incidence which occurred in a 29-dayculture from a 12-day embryo chick pancreas overa 96-hour interval is recorded in Figures 34 through38. The tubule P which had commenced to unwindat both ends (F1, upper extremity; P2, lower extremity) in Figure 34 is shown after 18 hours inFigure 35. At this time it had been broken by thelocal cells as documented by a 16-mm. time-lapsemovie sequence.' The two fragments remained inthe same microscopic field for the first 24 hours(Figs. 36, 37) but after 96 hours they had becomeseparated by two microscopic fields of view. Theupper end (P,) which had been broken off as aspiral element had unwound further (Fig. 38a),whereas, the tubule or lower portion of the complex(P2) showed considerable elongation (Fig. 38b).

On only one occasion was a crystalline form ohserved to be completely contained within a cell.In this instance, a malleable ribbon-like crystal (Q)was located in a multinucleated giant cell (Figs.39, 40) of a 56-day culture derived from a 12-dayembryo chick pancreas. These photographs weretaken at a 2-hour interval and show the rapidchange in cell contour and malleability of the enclosed crystalline form. A time-lapse cinematographic recording' of this cell showed the crystalto protrude sharply at one point and then to return to a position entirely within the cell.

A series of hexagonal and rhomboidal plates areshown in Figures 41 through 44. One cell is shownnear crystals in Figure 41. Careful inspection ofthe micrographs indicated the many layers ofcrystals and the stairstep pattern of growth. Angular measurements are indicated in the legend.

Cultures constructed with strips of cellophanehave never produced crystalline or particulateforms. Similarly, the cultures constructed with cellophane sheets containing the four needle holes didnot give rise to these elements.

MICROCHEMICAL RESPONSES

Megaparticles did not react positively to anyof the conventional procedures of cytochemicalanalysis listed in the section “Materials and Meth

1 A 16-mm. time-lapse film was shown at the Thirteenth

Annual Meeting of the Tissue Culture Association, Washington, D.C., May 59, 30, and 31, 1965.

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RosE—Biologic Particks in Ti@isue Culture 9283

forms. An abundance of filamentous forms weremore frequently observed in cultures of the centralnervous system.

The most prominent of all particles were thetubules, which proceeded through a transformation to tightly wound spirals, to loosely coiledspirals, to gently folded ribbons, and frequently tostraight ribbons. The spirals were always dextrohelical or produced a right-handed threading. Themalleability of the spirals (strength), snap-back ofthe tubules (elasticity) after having been bowed,uncoiling procedures, and occasional double heliceswere unique characteristics.

Observations of the developing forms denotetwo basic points : (a) the dialysis compartmentwith living cells was required to produce the illustrated particles and crystals and (b) living cellswere not observed to give rise to the crystals. Thesecond part appears to exclude the first; however,cells have been observed in the living form byphase contrast (7, 27, 28) and in the fixed form byelectron microscopy (3, 9, 27, 28) to contain crystals, presumably to give rise to them (27). Particlesin this report were observed to enlarge extracellularly in the microscopic range of size. It is not,therefore, unreasonable to consider that their ongin in the submicroscopic realm was first intracellular and that growth continued extracellularlyowing to the barricading effect of the cellophanewhich prevented their passage to the nutrient compartment; when their size reached 0.25—OSjs orbetter they were observed optically. Further, thisculture environment was highly desirable for themaintenance of differentiating embryonic tissue,and the particulate and crystalline formation apparently did not in any way adversely affect thiscellular growth. As a matter of fact, this differentiated growth was, in all probability, responsiblefor their production, which, because they couldnot leave the environment, simply interlocked andgrew in physical dimensions from submicroscopicto microscopic sizes.

Further, it appeared that those cultures withthe most differentiated growth produced the largest number of megaparticles and crystalline forms,whereas cultures composed exclusively of fibroblasts were totally devoid of them. Since thesemegaparticles and crystals gave positive responsesto RNA and protein staining, it seems probablethat they are protein products of the cultivatedcells and visible evidence that these cells are carrying out their differentiated functioning capacity ofsecretion. The elongating growth of tubules andspirals in uniform dimensions, their malleability,and distortion by heat and alkali suggested a poly

ods.― However, fixation with the potassium permanganate solution apparently altered the molecular structure of the helical forms sufficiently topermit an interaction with the pyronin (pH 5).Helices, ribbons, hexagonals, and rhomboidal formsall stained pink but failed to do so after 1 hourof digestion in a solution of ribonuclease. The organic solvents (methanol, ethanol, and chloroform)effectively dissolved all megaparticles, and definitealterations by heat were recorded. Hexagonal andrhomboidal crystals commenced to show a rounding of angular points and a distortion of straightsides at 80°C., whereas, the tubules and heliceswere markedly distorted at this temperature. Water replacement immediately (less than 1 minute)lysed the cells without evident changes to themegaparticles over a 924-hour observation. HC1produced an immediate fixation of the tissues anda precipitation of the fluid in the culture environment without any obvious effect on the megapartides. On the other hand, the response to NaOHwas an immediate lysis of the cells and a slow (15min. to 2 hr.) destruction of the megaparticles.The disturbance to the megaparticles produced byheat and NaOH was similar. The effect of NaCIwas not apparent after a 24-hour exposure. Toluidine blue solutions stained helical particles whichhad been fragmented by short exposures to NaOH(1/M) metachromatically (red) and was further

evidence for their protein quality.

DISCUSSIONForty-four micrographs have been used to illus

trate a variety of particulate elements produced bycultures of embryo chicks isolated under dialysiscellophane screens on coverslips of multipurposeculture chambers. The origin of these forms, theirdetailed biochemical identity, and usefulness to theculture environments in which they were foundremain obscure though temptingly speculative atthis time.

Needle-like crystals were observed as early as 8days after cultivation commenced, but generally92—3weeks elapsed before defined elements could beperceived easily by lOX objective microscopicscanning. Particulate growth was slow yet persistent. Rhomboids followed other rhomboids inspecific areas, but so slowly that time-lapse cinematography at one frame per minute failed to detect a growth rate. The highly refractile hexagonalcrystals, as shown in Figure 3, were usually a lateelement and appeared to be built upon other tubesor ribbons. Larger hexagonals (Figs. 41 through42) were also late-appearing and occasionally appeared to be built upon ribbons and filamentous

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Cancer Research Vol. 923, February 19639284

meric construction, whereas the layered growth ofrhomboids and hexagonals, their rigid forms, andmelting response to heat and alkali suggested atrue crystalline structure. Further, the resistanceof all forms to the conventional cytochemical technics was indicative of their homogeneous solidstate. Furthermore, formalin fixation is not considened a good fixative for nucleic acids, since itblocks a large number of the reactive groups toboth basic and acid dyes.

As indicated by the title, this report is simplyan introduction into a new field of study and admittedly is far from complete. For instance, it isnot certain that all the particulate and crystallineforms have been identified morphologically andthe sequences of transformations have only partially been followed. Their precise solubilities, responses to enzymes, chemistry, and molecular patterns as revealed by electron microscopy and x-raydiffraction are some of the essential analyses required to determine specific biostructural identity.Obviously, the tubular forms were not biochemically the same as the ribbons, nor were the rhomboids the same as the hexagonals; yet there wereinterrelationships between these variations whichsuggested biosynthetic processes at a magnifiedlevel.

Human embryo and neoplastic tissue culturessimilarly are being evaluated for their megaparticulate productivity. Recent experiments havedisplayed their capacity to give rise to similarforms.

ACKNOWLEDGMENTS

The author wishes to express his gratitude to Dr. Felix L.Haas and to Dr. C. M. Pomerat, who critically reviewed themanuscript and offered helpful suggestions; and to Mrs.Katherine Peterson who prepared the tissue cultures.

REFERENCES

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S. EPSTEIN, M. A. Some Unusual Features of Fine StructureObserved in HeLa Cells. J. Biophys. Biochem. Cytol.,10:153—OS,1961.

4. FULLMER, H. M., and Ln.uE, H. D. Dilute UnmordantedHematoxylin as a Stain for Basic Nuclear Protein.“Abstr.―First Annual Meeting of the American Societyfor Cell Biology, Chicago, 1961.

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8. GURR, E. In: Methods of Analytical Histology and Histochemistry, pp. 134—35, 153—54. Baltimore: Williams &Wilkins Co., 1959.

9. HADEK, R., and Swwr, H. A Crystalloid Inclusion in theRabbit Blastocyst. J. Biophys. Biochem. CytoL, 8:836—41,1960.

10. Hyrciixxss, R. D. A Microchemical Reaction Resulting inthe Staining of Polysaccharide Structures in Fixed TissuePreparations. Arch. Biochem., 16: 131—45,1948.

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55. Ross, G. G., and STEHLIN, J. S. The Golgi Complex andMelanin Elaboration of Human Melanomas in Tissue Culture. Cancer lIes., 21:1455—60, 1961.

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PLATES 1—10.—Abbreviations.

81),spiral.ri, ribbon.

In,tubule.

rh, rhomboid.lie, hexagonal.fi, uilamentousparticles.cx,explant.Ib,fibroblast.

ma, macropliage.

Na, nucleus.gi, globular images within and exterior to tubes.A, 13, C, etc—specifIc particles and crystals mentioned in text.1, @,.@,etc—specific areas of spirals at two levels of focus (Figs.

@20,51).a,b,c,etc—specificanglesinrhomboidsandhexagonals.iii, high microscopic focus.@1ii,mid-microscopic focus.Lo, low nlicroscopic focus.Oh, 18/i, etc.—initial photograph (zero time), 18 hours after

Oh,etc.Od,16d,etc—initialphotograph(zerotime), 16daysafterOd,

etc.

FIGS. 1—4.—Low-power phase-contrast inicrographs of outgrowths from a 15-day embryo chick cerebrum after 58 daysunder a full sheet of cellophane in a multipurpose culture chainher. A magnification line for Figs. 1-4 is in Fig. 4. X 180.

FIG. 1.—Spirals (sp) and ribbons (ri) peripheral to thecerebral explant (ex).

FIG. 5.—Numerous overlapping rhomboid (rh) crystallineforms next to the cerebral explant (ex).

FIG. 3.—A cluster of highly refractile hexagonal (he) crystalline forms accumulated in a nestlike space of cerebral cells.Hexagonal crystals of this smaller and denser variety were notalways a perfect six-sided form, but usually one end was perfectly pointed, sometimes both ends. (See also Figs. 41 and 45).

FIG. 4.—Filamentous forms (fl) with gentle sweeping andcircular contours adjacent to the cerebral explant (ex).

FIGS. 5—9.—Low- and high-power phase-contrast micrographs of tubular (tu) elements. A magnification line is in Fig. 6for Figs. 6—9.

FIG. 5.—Many tubular (tu) elements at the periphery of a19-day embryo chick gonad explant (ex) after 56 days of cultivation. A large fibroblastoid (fb) cell serves as a good index ofthe particulate size and population density in some areas.x500.

FIG. 6.—High-power phase-contrast micrograph of a clusterof tubular (In) elements observed in a 35-day culture of 15-dayembryo chick proventriculus. X 1800.

FIG. 7.—High-power phase-contrast micrograph of a macrophage passing over a tubule (tu) in a 58-day culture of 15-dayembryo chick pancreas. Arrows indicate extension processesformed i)y the macrophage along the tubule as it passed over it.X1800.

FIG. 8.—High-power phase-contrast micrograph of a tubule(tu) observed in a 45-day culture of 15-day embryo chick pancreas showing granular elements aligned along the surface ofthe tube. This was a common observation on old particles inareas in @vhichthe cellophane and glass coverslips were closelyapposed. X1800.

FIG. 9.—High-power phase-contrast micrograph of a tubule(In) from a 45-day culture of 15-day embryo chick pancreasshowing a sharp angular bend. X 1800.

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FIGS. 10—15.—Medium-power phase-contrast micrographsof helical and ribbonlike spirals from the cerebral culture shownin Figs. 1—4.A magnification line is in Fig. 10 for Figs. 10, 11.X360.

Fios. 10 and 11.—The specific area shown in Fig. 1 is reproduced at higher power in Fig. 10. Particulate elements A, B,and C are indicated. In Fig. 11 the same area is again shownafter 76 days of cultivation or after an 18-day lapse of timebetween the two micrographs. The helical forms A and B wereenlarged and elongated, and the division of the partitions ofribbon C was considerably elongated. Spiral A appeared to becomposed of two adjacent ribbons.

FIG. 15.—A rather long helix (D) was in a semi-unwoundform through its mid-section. Another helical form (E) was

loosely coiled. Other unlabeled helices and ribbons are shown.X535.

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FIG. 13.—Low-power bright-field micrograph of helicalforms derived from a 65-day culture of 11-day embryo chickintestine. These helices were weakly stained in the chamberwith hematoxylin after formol fixation. X733.

FIGS. 14—16.—High-power bright-field micrographs ofhematoxylin-stained helical spirals from the culture shown inFig. 13. In Fig. 14 the helix F is shown with a secondary helix Gon its right-hand portion. The helix F was observed to be

denser in its left extremity, suggesting that helix G split fromthe right extremity of F. A similar record is shown of the helicalform in Fig. 15, and in Fig. 16 there is a suggestion of a triplingof helical forms. X 1500.

FIG. 17.—High-power phase-contrast micrograph of twinhelices which appeared to have resulted from a longitudinaldivisioning. This was derived from a 64-day culture of 11-dayembryo chick cerebrum. X 1535.

FIG. 18.—A low-power phase-contrast micrograph of ahelical form which had divided at its right-hand extremity toform two antennalike components. This helix was derivedfrom a 00-day culture of 11-day embryo chick cerebrum. Alesser-sized helix is shown crossing the antennalike ends.X360.

FIG. 19.—A tubular form which had unwound to form ahelix and possibly to give rise to a secondary helix. This partide was observed in a 73-day culture of an 11-day embryochick lung. X 1900.

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FIGS. 50—53.—High-power phase-contrast micrographs oftwo tubules observed in a 50-day culture of a 15-day embryochick pancreas. The magnification line is shown in Fig. 55 forFigs. 50—53.X3400.

FIGS. 50 and 55.—High-focal level micrographs (Iii) showing the striping which occurred on the surface of the tubes.In Fig. 55 a helical unwinding occurred at both extremities.Specific spirals in Fig. 50 are numbered.

FIGS. 51 and 53.—Mid-focus micrographs (Mi) of thespirals of Figs. 50 and 55 showing the prominent dashing produced by the forming spirals. This was particularly evident inthe tube in Fig. 51, delineated by the numbered arrows whichmatched the Ilumbered spirals in Fig. 50.

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FIG. 54.—Low-power phase-contrast micrograph of a helixwhich had been distorted so that a single, though symmetrical,loop was formed. This was observed in a 60-day culture of a15-day embryo chick pancreas. At the top of the loop there isa nonrefractile hexagonal-forming crystal. X560.

FIGS. 55—57.—High-power phase-contrast micrographs of ahelix which surrounded a tubule taken at three focal levels(high, iii; mid, Mi; low, La). X5700.

FIG. 58.—Three tubular forms (II, I, J) are shown withpartially unwound spiraling (sp) extremities. The tubule I!also had a sharp elbow bend and globular (gi) internal andexternal images. X 1650.

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FIG. 59.—High-power phase-contrast micrograph of a broadunspiraling portion of tube K. A lesser-sized tubule (L) withencircling helical images and several other tubes were in thisfield. X 1600.

FIGS. 30, 31.—Two medium-power phase-contrast micrographs of a tube which was bowed after the coverslip of theculture chamber was gently wiped with lens tissue for cleansingpurposes. Within a few minutes this bowed helix had returnedto the straight form. X600.

FIG. 35.—High-power phase-contrast micrograph of twotubes, .1! and N. Tube N apparently surrounded tube if. Theunspiraling which occurred in the upper extremity of N produced the encircling helical form similar to that of Figs. @5—57.A portion of a large spiral 0 is also shown. X1700.

FIG. 33.—High-power phase-contrast micrograph of thetubule .11 encircled by the helical extremity of N. X5750.

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FIGS. 34-38.—Medium-power phase-contrast micrographsof a tubule-spiral complex (P1, P2) observed in a 50-day cultureof a 15-day embryo chick pancreas. Numbers in the lower leftportion of each illustration indicated the hours between micrographs. A magnification line for Figs. 34—38is shown in Fig. 34.X750.

FIG. 34.—Complex (P,, P2) as it was positioned betweentwo cellular elements.

FIGS. 35 and 36.—During the first 18 hours the tubule-spiralcomplex was separated into a spiral component P1 and a tubular component P2 by the cellular activity.

FIG. 37.—After 54 hours the cellular elements separated thecomponents Pi and P2.

FIG. 38.—By 96 hours the two components were so widelyseparated that two separate micrographs were made (Figs. 38aand 38b). Pi is shown in Fig. 38a to have uncoiled and elongated 54 times its original length as shown in Fig. 37. Crystalline element P, is shown in Fig. 38b to have elongated to agreater dimension than the original complex P1, P, shown inFig. 34.

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FIGS. 39, 40.—High-power phase-contrast micrographs of amacrophagic multinucleated cell containing an elongated partide (Q) of uncertain type. This was observed in a 56-day cul

ture of a 15-day embryo chick pancreas. The change in contourof the particle during the 5 hours separating the micrographs

denoted its malleable quality. The magnification line is in Fig.39 for Figs. 39—40.X5700.

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FIGS. 4 1—44.—High-power phase-contrast Ilucrographs ofhexagonal (he) and rhomboid (rh) crystalline elements. A magIlification line is in Fig. 44 for Figs. 41 through 44. X 1735.

FIGS. 41, 45.—Hexagonal forms from a 65-day culture of a13-day embryo chick pancreas. Angles a (107°)and b (156.5°)are labeled. All hexagonal crystals were formed of these angular

measurements.

FIGS. 43, 44.—Rhonlboids formed by angles c (80°) and d(100°)from a 355-day culture of an 11-day embryo chick lung.Note the many forming layers.

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1963;23:279-284. Cancer Res   George G. Rose  IntroductionBiologic Crystals and Particles Produced in Tissue Culture: I.

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