NASA Technical Reports Server (NTRS) 19970026255: Feeding Frequency Affects Cultured Rat Pituitary Cells in Low Gravity

24 Pages·1.4 MB·English

by  NASA Technical Reports Server (NTRS)

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ELSEVIER Journal of Biotechnoio.,zy 47 11996) 289-312
Feeding frequency affects cultured rat pituitary cells in low
gravity
W.C. Hymen*, R.E. Grindeland b, T. Salada _. R. Cenci", K. Krishnan", C. Mukai c,
S. Nagaeka _
"Dcparzment vf Biochemi._trr and tl( lecular Bioh)7.y. The Pc,n,',::_::J State L)mcr._ity. l_'nivcr_ity Park. P.4 16S02. US.4
_.V.-IS4 .4rues Research Center. I1,.!:_:t Ficld. C.4 940-75. USA
_.Vational Space Development 4'.'cpw.:' c:tJapan. lhtaraki. 305. Japan
Received 2 October 1995: revised IS December i995: accepted 23 December 1995
Abstract
In this report. _e deacribe the results of a rat pituitary ceil c=!:ure experiment done on STS-65 in which the effect
of cell feeding on the release of ,.he six anterior pituitary. ho,.,'rne,nes _as studied. We found complex micregra_ ity-re-
fated interactions between the frequency' of cell feeding and _hequantity and quality' (i.e. biological activit.vl of some
of the six hormones released in flight. Analyses of gro_th hormone IGHI released from cells into culture media on
different mission day's using gel filtration and ion exchange chromatography yielded qualitatixely similar results
betxveen ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight tbut not ground)
cultures. Vigorous fibroblast grot_th occurred in both ground and flight cultures fed 4 times. These results are
interpreted within the comext of autoc,'-ine and or paracrine feedback interactions. Finally', the payload specialist
successfully' prepared a fresh trypsin solution in microgravit._, detached the cells from their surface and reinserted
them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In
summary, this experiment shows that pituitary.' cells are microgravity sensitive and that coupled operations routinely'
associated with laboratory cell culture can also be accomplished in low gravity.
Keywords: Rat pituitary cell culture: Microgravity
1. Introduction some time. Many different types of cells have
been exposed to low gravity; some show signifi-
If and how cells sense microgravity has been cant and repeatable changes (Halstead et al.,
thoughtfully considered, modeled and debated for 1991; de Groot et al., 1990; de Groot et al., 1991;
Limouse et al., 1991; Cogoli, 1993).
*Corresponding author. Department of Biochemistry. and Our group has studied rat anterior pituitary
Molecular Biology, The Pennsylvania State University, Uni- gland cell structure and function during and after
versity Park, PA 16B03, USA
spaceflight (Hymer et al., 1992; Grindeland et al.,
Presented in part at the American Society of Gravitational
1987). We have focused on synthesis and release
and Space Biology Meetings, Washington, DC 1995.
0168-1656_96, S15.00 0 1996 Elsevier Science B.V. All rights reserved
PII SO168-1656(96)01373-9
290 It'.C.
H)mer ctal. Journal of Bioze_'lmolog.v 47 (1996) 289-312
of growth hormone (GH) and prolactin (PRL) into a single cell suspension that routinely yields
molecules because these two protein hormones are 2-2.5 x 106 cells from each gland (Hymer and
known to participate in the regulation of muscu- Hatfield, 1983). For this experiment, total cells
loskeletal, immune, vascular, metabolic and en- recovered were 4.8 x 108 with a viability > 95%.
docrine systems; systems which are often changed As shown in Fig. 1, 4 x 107 cells were seeded
in low gravity. We recently reported that pituitary into each of six cell culture kits (CCK) specifically
cells, in a passive cell culture system in low grav- designed for cell culture technology in micrograv-
ity, show differences in the quantity and quality ity. Three CCK's were maintained in an incubator
(bioactivity) of GH and PRL released from pri- designed for spaceflight operations on Shuttle
mary rat pituitary cells in vitro (Hymer et al., middeck while the other three were kept under
1996a: H.vmer et al.. 1996b). Interestingly. some similar conditions in the laboratory at Kennedy
of these changes were similar to those found in Space Center. Constant real time monitoring of
pituitary cells prepared from spaceflo_n rats after operational parameters of this incubator in micro-
7-14 days in microgravity. gravity indicated that the temperature variance
The unique design of the cell culture hardware between the ground and flight units was __.0.1°C:
available for this experiment permitted us to relative humidity ranged between 40-60% in both
study, for the first time, possible effects of cell units throughout this 17-day experiment. The cul-
feeding on hormone release from each of the six ture medium (15 ml CCK) was identical to that
major hormone-containing cell types. It also pro- used in a previous rat pituitary microgravity cell
vided the astronaut the opportunity [o prepare culture experiment rhymer et al., 1996a): viz.
fresh solutions from preweighed powders and use modified Eagle's minimum essential medium
them to trypsinize the anchorage dependent pitu- (:_ME.'vll containing 5% calf serum. 0.2%
itary cells from their surface. NaHCO3, 25 mM Hepes buffer (pH 7.4) and
This experSment not only demonstrated that antibiotics.
coupled technologies routinely associated with cell Culture media in CCK #1 and #2 were
culture procedures on earth could be accom- changed according to the schedule shown in Fig.
plished in the unique low gravity environment, 1:cells in CCK # 3were left undisturbed over the
but also showed that important microgravity entire course of the 17-day experiment.
changes in the quantity and quality of certain
hormones released from this mixed cell culture 2.2. Cell culture hardware.
occurred. In some cases, these changes were posi-
tively correlated with the frequency of cell feeding The unique design of the CCK and its associ-
during 14 da._s in space. ated hardware permitted (a) microscopic obsera'a-
tion of the primary rat pituitary cells attached to
the pronectin-treated polycarbonate surface; (b)
2..Materials and methods removal of spent media and their storage at -
20°C; (c) addition of fresh culture media; and (d)
2.1. Anflnals and tissue processing before flight on board preparation of two fresh solutions from
preweighed powders stored in syringes (10 mg
Carl and use of animals for this experiment Difco 1:250 crude trypsin and 100 pg DNase
that was done on the Space Shuttle in 1994 (STS- (Type I, Sim'na)).
65) was approved by IACUC committees at Penn Results of numerous preflight trials using pitu-
State and NASA Ames Research Center and con- itary cells in CCK's indicated that pretreatment
formed to NIH guidelines. Three days before with pronectin (10 /_glml, Si_ana) significantly
launch, 150 specific pathogen-free Sprague Daw- helped promote cell attachment and also en-
Icy male rats (200-220 gms, Harlan Sprague hanced cell recovery after trypsin (data not
Dawley, Frederick, MD) were killed by decapita- shown). The appearance of a CCK and its opera-
tion and their anterior pituitary glands dissociated tional configuration is given in Fig. 2. Basically,
H".C.Hjmcr st al. ,,Journal o.[Btot(,(hmdot,l"47(1996_289-312 291
150 Male Rats
Anterior PituitaryGlands
T_psinize
4.8 x 10acells
i
Y
seed in 6 cell
culture kits(CCK)
3days before launch
[4x 107cetls/CCK]
/\
3 ground 3 space
(37"C; c5% air (37°; 95% air
5% CO.-) 5% C02)
Media change
sohec_;e
T
I i I I I I i I
(Lo3) 2 4 6 8 10 12 14
4 Mission Day
CCK#1 X X X X
CCK#2 X
CCK #3 X
+Trypsinized and replated
Post'flightAnalysis:
HPLC Frozen Media Ce,s
Gel filtration • hormone immunoassay • flow cytometry
ion exchange • (GH, PRL, LH, FSH, TSH, ACTH) • image analysis
• hormone bioassays
• immunocytochemistr_,
• (GH, PRL)
Fig. 1.Experimental design. See Materials and methods for details.
2_2 II (" H'mtr ,t .d. J,,urnul of Bu_technoh,gy 47(1996) __g¢9-.,1"_ "
this unit con:d_,ts of II) a polycarbonate sheet tion effectively breaks down nucleoprotein that
(0.5-mm thick)to xxhich the anchorztge-dcpendent may have been released from dead cells and rou-
pituitary ce!ls attach and 12) mo chambers tinely results in a smooth suspension of single
bounded bxa 3.0-a NucleoFore metnbrane which cells (Hymer and Hatfield, 1983). After with-
permits x_i',hdr:mal and addition of culture drawal of the trypsin-trypsin inhibitor solution
medium from the chamber distal to the attached into a storage syringe, the cells were reinjected
into CCK #2 and 15 ml of fresh culture medium
cells. The trypsin solution ._as prepared by pass-
added for continued culture (Fig. 1).
ing 10 ml of a 5 mM Hepes buffered solution
Icontaining 30 m>.l glycine, cl.2 mM potassium
2.3. Poszflight uriah'sis
acetate. 0.3 taXI .MgCI:6H-O. 0.03 mM CaC],.
220 mM glycerol, a.a m.Xl sucrose and 0.2 mM
Within 3 h of Shuttle landing, cells in each of
ZnC1, and 0.2'. EDTA I. pH -."6 into the syringe
the six CCK's were {a) photographed: _b) media
containing pox_dcred Ir:...-.,i:l Icoupled luer
withdrawn: (c} cells removed from CCK # 1 and
filtingst io el'l'cct >olution. \:"er Hue removal of
# 3 by trypsinization and (d) cells in CCK # 2
cuhure medium. _his solu'{cn *._asthen added to
osmotically lysed by addition of dH.O containing
the attached cells in CCK =2 i10 mini to pro-
0.2 mM ZnCI,. The viabilities of cells removed
mote d,t,._ ....,,nt _monitore_ :v.{croscopically) fol-
from CCK #1 and #3 were >80%. A portion
lowed by add:Aion of 5 mi ,'f., so, bean trypsin
(1 x 10") of these cells were used for morpholog-
inhibitor <clu;ion (1 nagn:'._ teat had been pre-
ical analysis lsee below): the remainder subjected
pared in He?es buflk'r labo'.e_ before launch. The
to separation by' free flow electrophoresis. Results
cells were ihcn ,._ilhdrax_n :r:." :he CCK into the
from this later effort, as well as results of the
syringe comaming pox_dcre2 DNAase: this solu-
electrophoretic separation trials of lysed cells, is
published in the companion paper.
2.4..Uorpho/ogy
Cells in the initial suspension, as well as those
recovered at the end of the 17 day-culture period.
were either (a) fixed and processed for immunocy-
tochemical analysis of growth hormone (GH) and
prolactin (PRLI cells or (by multiparammer flow
cytometric analyses exactly according to our es-
tablished procedures that are described in several
previous publications (Hatfield and Hymer, 1985;
Perez et al.. 1993). Parameters evaluated by flow
cytometry {on 10000 cells/sample) were (1) GH
cell distributions: (2) marker indices, i.e. the ratio
of the voltages of stained to unstained cells _ an
index of "'brightness" of GH-specific fluorescence
staining: (3) forward angle light scatter, FALS.
(an index of cell size) and (4) perpendicular light
scatter, PLS, (an indicator of content of cytoplas-
Fig. 2. Cell cuhure kit ICCK) used in this experiment. Syringes mic hormone-containing secretory granules). Pro-
_ith luer fittings {.-_ enable media _i,hdru_al as aell as cedures for the immunocytochemical analyses of
addition of ne_ media: the actual culture chamber with its
GH stained cells attached to poly-I-lysine coated
metal casing removed is also sho_n _B_. See Materials and
cover slips were exactly as described previously
methods for additional details. (Phetographs copied with ap-
(Hatfield and Hymer, 1985); viz. fixation in Zam-
proval from NASDA; taken from IML-2 brochurel.
I|'.C. H:mer t't al. JourlTal of Bit te_hm,hn,'.v 47 (1996) 259-312 293
boni's fluid followed by membrane permeabiliza- Kidney Disease and the National Hormone and
tion with 0.4';',, Triton-x-10(): incubation in GH- Pituitary Program (University of Maryland School
of Medicinel.
specific polyclonal antiserum follmved by
Concentrations of biologically-active GH (bGH)
secondary amplification with horse radish peroxi-
dase antiserum. These cell preparations were used in culture media and extracts were determined
for digital analysis of the cytoplasmic area occu- exactly according to the tibiai line bioassay proce-
dure of Greenspan etal. {Greenspan et al., 1949).
pied by GH using procedures and equipment iden-
tical to those used previously (H.vmer et al.. 1992). Approximately 200 hypophysectomized female
rats. 26 days old at surgery, were used to assay
2.5. HPLC
samples using a four-point assay procedure (i.e. 4
rats dose2 doses). The assay endpoint measures
Txvo different types of chromatograph.', were
increases in tibial epiphyseal plate x_idths after four
done to analFze the molecular nature of the im-
daily injections of hormone: it has a sensitivity of
munoreactixe GH (iGHI contained in the culture
1 .,g and is specific for GH. Responses were
media taken from flight and ground CCK # 1.
compared to a bovine GH standard _1.5 U rag)
These were gel filtration chromalography to esti- calibrated against a USP standard: they are ex-
mate apparent molecular weigh',s of released iGH
pressed in terms of an in-house preparation of rat
IH.vmer et al.. 1996at and anion exchange chro-
GH 13.0 IU mg).
matograph.v to evaluate oxeralI charge character of
the released iGH. Samples from CCK _ 2and # 3
_ere not analyzed. In all cases, l-ml aliquots of 2.- Pro/a_ziir ¢PRL)
culture media were 1.vophilized. and reconstitmcd
in either 500 ,I of 0.1 M potassium phosphate PRL erlzyme immunoassay was done exactly as
buffer containing 0.05 M NaCI. pH 7.8 lgel filtra- de:cribed prexiousl.x ISignorella and Hxmer. 1984)
tion) or 250 :¢1of 200 mM TrisHcl. pH 7.8 for using polxclonal an'Aserum (cross reactixity to GH
< 0.3'";, at a dilution of 1:40000). Each sample was
anion exchange chromatography.
For gel filtration, each sample was applied to a analyzed in duplicate at two dilutions and the
column of Protein-Pak SW (7.8 mmx 300 mm. results are expressed relatix e to a rat PRL standard
Waters. Milford. MA) equilibrated with the same (B-7) kindly provided by the National Institute of
buffer. The column flow rate was 0.3 ml rain. The Diabetes. Digestive and Kidney Disease and the
column was calibrated with blue dextran (2 000 000 National Hormone and Pituitary Program (Uni-
MW). beta-amylase (200 000 MW). bovine serum versity of Maryland School of Medicine).
albumin (66 000 MW). carbonic anh.vdrase (29 000 PRL bioassay was done by the Nb-2 Ivmphoma
MW) and ribonuclease (13 683 MW). The flow rate cell assay originally described by Tanaka et al.
of the ion exchange column was 0.6 ml, min and (Tanaka et al., 19S0) and used routinely in our
used a 0.6 M NaCI gradient for elution. laboratory (Hymer et al., 1996b). This cell culture
bioassay is specific for PRL and has a sensitivity of
2.6. Hormone assays. Growth Hormone (GH)
0.2 ngml. It is based on the ability of PRL to cause
division in a line of T-lymphocytes prepared from
Concentrations of immunoreactive GH (iGH)
lymphomas of estrogenized rats.
released from cells into culture media were deter-
mined by enzyme immunoassay (Farrington and
Hymer, 1987). The polyclonal antiserum to GH 2.8. Follicle sthmdathrg hormone (FSH).
has a cross reactivity of < 0.3% to prolactin at the lutehffzbTg hormone (LH), thyroid stbnulatbTg
final dilution (i:80000) used in the assay; each hormone (TSH) and adrenocorticotropic hormone
sample was analyzed in duplicate at two dilutions (.4CTH) assa)'s
and results are expressed relative to a rat GH
standard preparation (B-l l) kindly provided by Radioimmunoassay kits for each of these pitu-
the National Institute of Diabetes. Digestive, and itary hormones were kindly supplied by the Na-
294 WC. Hymer el al./Journal of Biotechnolog)'47 (19961289-312
tional Institutes of Diabetes, Digestive, and Kid- in total ACTH released from unfed cells or cells
ney Disease and the National Hormone and Pitu- fed 4 x (Fig. 3F).
itary Program (University of Maryland School of
Medicine). All hormones were iodinated using 3.1.1. CCK # I
IODO-GEN _Pierce Chemical, Rockville, IL): 2.5 The kinetics of hormone release from cells fed 4
pg of purified hormone and 250 /iCi of NaJ::l. times sometimes revealed very different patterns
Assay protocols were exactly as described in in microgravity that were often dependent on
NIDDK technical notes. The following antisera feeding frequency and hormone type. For exam-
and reference preparations were used: (1) rTSH; ple. feeding cells 4 times during spaceflight re-
iodination preparation rTSH-I-9 (AFP-1308C), sulted in approximately linear increases in rates of
reference preparation rTSH-RP-2 (AFP-5153B), release of PRL IFig. 4D), FSH (Fig. 5A) and
antisera anti-rTSH (C21381); (2)rLH: iodination ACTH (Fig. 5G): hoxvever, the rates of release of
preparation rill-l-7 (AFP-9-104BL reference PRL. FSH and ACTH from corresponding
preparation rLH-RP-3 (AFP-7187B). antisera anti ground control cultures was different in each case.
rLH-S-10: (3) rFSH: iodination preparation On the other hand. the rates of release of GH
rFSH-I-$ (AFP-II454B). reference preparation lFig. 4AI and TSH (Fig. 4G) were similar between
rRP-2 (AFP-4621B), antisera anti rFSH-S-11 ground and flight. LH release IFig. 5D) was ini-
(AFP-CO972SSl: (4) hACTH used in het- tially 2-3 x more from flight cells, but later in
erologous assay (rat reagent not available): iodi- the mission these rates were the same as from
nation and reference preparation (AFP-6328031). ground cells.
antisera (AFP-2938C).
3.1.2. CCK # 2
These cells had their first medium change on
3. Results day 9 follo_xcd by an immediate tr.vpsinization
procedure x_hich resulted in cell detachment from
3.1. Dr vitro hormone release in microgravity the polvcarbonate surface (based on microscopic
observation in both ground and flight chambers).
The total quantity of immunoreactive GH, These were reinserted back into the same chamber
PRL, TSH, FSH, LH and ACTH released from and the culture alloxved to continue for an addi-
the three different cell culture kits during 14 days tional 5 days. In every case, the rate of release of
in microgravit.v, relative to synchronous ground each of the six hormones was greater from ground
control cells, depended upon the frequency of cells after trs'psinization (Fig. 4B, E and H; Fig.
media change and the particular hormone being 5B, E and H). However, in flight this release
measured (Fig. 3). Sometimes total hormone out- pattern was either similar (FSH, LH. TSH) or
put from both ground and flight cultures was opposite (ACTH. GH, PRL).
positively correlated with the frequency of media
change, whereas in other cases feeding frequency 3.1.3. CCK # 3
had little effect. The total amounts of hormone These cells were left undisturbed over the entire
released from either ground or flight cells varied experiment. The only changes in these flight cells
over a wide range: i.e. ~10 micrograms (LH, FSH, were (a) a 4 x increase in GH release (Fig. 4C)
TSH); 2-10 milligrams (PRL, GH); ~100 and (b) a 2 x increase in ACTH release (Fig. 5I).
nanograms (ACTH). Noteworthy fli_t-associated When these same media from CCK's # 1, 2
differences in total hormone release were (1) a and 3 were assayed for their content of biologi-
4 × increase in GH release from unfed flight cells cally active GH and PRL, interesting flight-re-
(Fig. 3A); (2) slight to moderate reductions in LH lated differences were found, both in total
release from fed cultures (Fig. 3E); (3) small in- amounts of bioactive hormone released and in the
creases in GH and PRL release from cells fed four kinetics of that release. In terms of total bGH
times (Fig. 3A and B); and (4) two-fold increases release, there was a -50% reduction from fed
W.C. ttymer t't u/. Journal of Biotechno/ogy 47 tl996; 289-312 295
12000 tO0
f(A) _GH
10000'
iJ _s)iP,I, BO
8000"
6O
!
co_ 6000'
,oi
E
4000,
20 "l
I
I I:'_"!! ' .!,':_._.. _'_-'I,-'_.
I
0 4
4 [ I 0
_5 300 I
(DI ,FS_, (F)_,:,CTH
i
i
,o j
200 .
, i:jli
E
1 o
Fig. 3. Total release of immunoreacdve (A) GH, (B). PRL, (C) TSH, (D) FSH, (E) LH and (F) ACTH from CCK # 1, # 2 and
# 3 in which culture media were changed 4, i and 0times, respectively, during 14days in microgravity. Note large difference in total
quantities of hormones secreted tmicrograms-nanograms).
flidat cells and a doubling of hormone from the by one-half (Fig. 6B). The kinetic data relating to
unfed flight cells (Fig. 6A). In terms of total release of bGH and bPRL indicated (a) a large
bPRL released, the situation was approximately burst in initial bGH from ground (but not flight)
reversed. Thus, there was a 2.5 x increase in total cells in CCK # l (Fig. 7A); (b) no correlation
bPRL release from cells fed 4 times in micrograv- between amounts of iGH and bGH measured in
ity while unfed PRL cells reduced bPRL output CCK # 3 flight media (cf. Fig. 4C vs. 7C); and
296 II".C. Hr•mer t't al. Journal of Bioteclm,h,g.r .,17(1996) _"89-. ;I."_
finally (c) a good correlation between iPRL and CCK=3
bPRL contents in CCK # I flight media (cf. Fig. .<._
ChanBes J_ Cha.n9e C_a__.
4D vs. 7D). (c}!
3.2. Anah'sis of released GH by HPLC
Each of the five samples obtained from CCK
i z_,
# 1, after fractionation by gel filtration chro-
matography, contained two peaks of iGH with
very different apparent molecular weights (Fig. 8). o
One of these approximated to the knoxvn mass of iL_leinizin_ Hormone {E)
GH. viz 22 kd. The other had an Jpparent molec- IC [] _!i_'-,t
ular weight > 2 × I0:. Neither coincided with
the OD 2S0 profile _hich represents the major
(¢)]
protein classes in the serum containing medium
I !
(Fig. 8. top). Generally speaking, the iGH elution I I ,I'-_J';:
profiles bet\_een the ground and flight samples
_5
i g)
CCK-'2 CC;::3 i I
I CEK-"I
C"ance Chzn;esl
50 t (A) ;mmuno'reaclive (i) {_} (C) g I
_"o ] Growth Hormone r-_,
_ OS r_.f._ .__:..
[]
¶'= 30'_]0 r: _ . I_,V;.r,::t;_.!11I :!
b 2°'I
rJ_ 2 5 8 11 14 2 5 8 11 ;4 9 14 9 14 14 't4
i.- Mission Day
I
Fig. 5. Kinetics of release of iFSH. iLH and iACTH from
a2
(E) (Flj CCK's I-3 on earth and in micro_ra_iL_.
T--
E
were similar, although flight samples tended to
have more low molecular weight hormone. Fi-
I
nally, there was a progressive loss of high molecu-
"c I
i lar weight iGH from both ground and flight
l_,-I I samples as the time of culture increased. Total
Ill
o
recoveries of iGH from both ground and flight
lOO
T {G) itSH (HI {i)
samples after this step averaged 46 + 3% (n =
I0). Of the recovered iGH, a majority was in the
lower molecular weight region (range 56-81%).
After fractionation by anion exchange chro-
matography, each of the ten samples from CCK
# 1contained a single peak of iGH which eluted
from the column before the salt gradient began. A
2 5 8 1114 5 8 11 14 g 14 g 14 14 14 majority of the OD 280 material also eluted in
MissionDay this same re,on (Fig. 9. top). While the amount
of iGH from the flight samples remained rela-
Fig. 4. Kinetics of release of iGH, iPRL and iTSH from
CCK's I-3 on earth and in microgravity. tively constant throughout the culture, there was
I1"('. Hi'mcr t't al. J,,urnal of Bi,tcHmuh,ey J7 (19061 289-312 297
a progressive loss of iGH from the ground sam-.
ples. The reason for this pattern is unknown. i CCK =1 CCK :2 I CCK _3 I
Ct_anges Chan_e I Changes I
Recoveries of iGH from both ground and flight
3{)WF" (A) Bioaclive (B) (el
samples (i.e. fractions 2. 3 and 4) after these steps
Growth Hormone
averaged 88 + 10% (n = 10) with no differences
between ground and flight samples. Also shown in 20' [] Groun_ [] Flight
Fig. 9 (right column) are the biological activities
of GH in fractions 2 and 3. assessed by the tibial
line assay. Sometimes the patterns of bGH paral-
leled those of iGH (e.g. day 2. 5 and 14): other
samples did not (e.g. day III. It is especially
_00 .{D) Bioacfive (E) (F)
interesting that the bGH concentrations were usu-
:_] Prclaclin
ally 2-7 x greater than those measured by im- -I mC:
munoassay. Recoveries of bGH from both ground
and flight samples averaged 373 -:- 80'11,tn = 10t
more than what was estimated to be present in the
original sample of culture medium. This high
recover}.' after ion exchange chromatography may i!!ni7!0
:-_i !! !/.I i-_,.!,/:,
result from removal of an inhibitor of bGH activ-
2 - 3 _ ;4 2 5 8 _1:4 9 14 9 :4 ',4 .4
ity contained in the unfractionated sample.
Mission Day
The different profiles of iGH obtained after gel
filtration and ion exchange chromatography Fi'.'. -. Kinetics of relea.-,e of bGH panels L-X-C) and bPRL
_?and: D-FI from the 6 CCK's. Note scale difference.
prompted us to do a single rechromatography
12000 (A) bGH,
trial in _hich iGH molecules in fractions 2 and 3
_cre subsequently fractionated on the sizing
10000 coiumn. Because supplies were limited, this trial
aas done only on the mission day 8 sample. The
results show that what iGH was recovered (31%
SO00
ground: 11% flight) had an apparent molecular
i
weight in the range of 22 kd: none of the high
_o molecular weight material in the original sample
6000
E IFig. 8) was detected in this region after rechro-
matography (Fig. 10).
4000
3.3. Cell morphology
2000
Phase contrast microscopy of cells in the flight
CCK's obtained within 3 h after Shuttle landing
showed that live (phase bright), attached cells
0
were present in all ground and flight chambers.
Considerable care and time was taken to pho-
Medll Chin;el tograph the cell growth and distribution patterns
in each CCK from random areas at several mar
Fig. 6. Total release of bioactive GH (A) and bioacti_e PRL
nifications. The photographs shown in Fig. 11 are
(B) from cells in CCK ¢ I- # 3 in _hich media were changed.
4. I and 0 times, respectively, during 14 days in microgavity. taken at two different magnifications: they docu-
298 W.C. H)mer et al. /Journal of Bioredmoh,gy 47 (1996) 289-312
ment attachment, cell viability, and growth pat-
Gel Fill r,$lion
2.0 o -- terns that are representative of the entire culture.
_ . ° u_
A.t low magnification, the dominant feature of
1.5 cells in CCK # 1 and # 3 was their clumping in
CCK # 1, both ground and flight (Fig. 11A and
B) and extensive clumping in flight CCK #3
1.o
(Fig. I1F) but not ground CCK #3 (Fig. liE).
Clumping of cells is easily seen because of scat-
O.S
tered light which renders the cell clumps dark in
the photomicrographs. Morphometry of these
clumped areas from 3 different frames (entire
photographic area) showed that these clumps oc-
...o--. FIiGhi cupied 10.9 ± 1.0%(CCK # I. ground): 12.6 ±
2.6% (CCK # I. flight): 6.7 ± 0.5% (CCK # 3.
, .,.,
ground); and 62.6 ± 4.4% (CCK # 3, flight) of
i1 MiSSiOn day 2 / ---"_ _roui'_d the total area. Cells in unclumped areas at higher
magnification showed epithelial cell morphology
that is typical for primary rat pituitary cultures
except for one feature; i.e. total absence of fibrob-
S : •
last cell growth in unfed cultures from both flight
4 " .
• .."7%¶ and ground samples (cf. Fig. I1G and H vs. IIC
B_l Mission day S I,. and D). Comparison of Fig. 11C vs. I1D suggests
that fibroblast growth is greater in microgravity.
but other photographs (not shown) do not. The
cell images in flight CCK # 2. after Shuttle land-
=-
ing, indicated that these anchorage dependent pi-
4 ::"b
• tuitary cells, after tr3"psinization on day 9,
reattached in microgravity sometime during the
last 5 days of the mission IFig. 12). These were
8 Mission day 11
more clumped than ground controls.
6 L
After Shuttle landing, our experimental design
4 required cell removal from CCK # 1and # 3 by
2 trypsinization in order to do both cell image
analyses as well as cell separation trials by contin-
0
8 uous flow electrophoresis. The results of the elec-
Mission day 14 (landing)
trophoresis trials are the subject of the companion
6
report. In Fig. 13, we show the general features of
4 .l° trypsinized cells prepared from CCK's #1 and
2 # 3 (both ground and flight) after immunocyto-
0 chemical staining with a GH-specific antiserum.
S 10 1S 20 25
At this level of discrimination, there were no
Fra_ion
obvious differences in GH cells (darker cells in
Fig.8. Fractionationof culturemedia from spaceflown and Fig. 13) or non-GH cells between any of the
ground controlcellsinCCK _ Iby gelfiltrationHPLC. The treatment groups except for a few (< 10%) "gi-
opticaldensity(OD 280) profileofserum proteinsintheday-8 ant" single cells (or remains thereof) in flight cells
flightsample isshown in the top panel together with the
from CCK # 3. Image analysis of immunocyto-
elutionpositionof standard molecular weight markers. OD
profilesofallothersamples were identical. chemically stained GH cells indicated that some