From the Department of Pharmaceutics and Biopharmaceutics, Christian-Albrechts-University3. Kiel, and Department of Pharmaceutics and Biopharmaceutics, Free University of Berlin*1, Berlin (Fed/Rep. of German)
Quantification of Cell Subpopulations, Fractions of Dead Cells and Debris in Cell Suspensions by Laser Diffractometry
By S. Rudta, T. Blunk3, and R. H . Mullerb
Summary
Laser diffractometry was employed for size analysis in liver cell and blood cell suspensions to assess its suitability for character- izing cell populations. The method proved sensitive to detect sub- populations in liver cells (bimodal or trimodal distributions) and to quantify their volume fractions. Cell debris and aggregates of cells could also be quantified, dead cell populations recognized by their shift in the mean cell diameter. Laser diffractometry is there- fore suitable for determining the quality of cell isolations (e.g. by liver perfusion) or for following alterations in cell populations during culture of cells in suspension. Analysis of human blood allowed differencial ions to be made between thrombocytes and other blood cells. No peak separation was obtained for the pop- ulations of erythrocytes and granulocytes due to their similarity in size. Monocytes could not be detected due to their extremely low number in the blood indicating the limit of the method.
Zusammenfassung
Quant ifizierung von Zcllsubpopulationen. Fraktionen von toten Zellen und Zelltriimmern in Zellsuspensionen mittels Laser-Dif- fraktometrie
Die Laser-Diffraktometrie wurde fur die Grofienanalysc 1,1 ^ berzell- und Blutzellsuspensionen eingesetzt. urn ihre Eig*nil * ^ Charakterisierung von Zellpopulationen zu untersuchen. L>i • thode erwies sich als ausreichend empfmdlich, um Subp°Pu y^r nen in Leberzellen zu detektieren (bimodale oder tritnoda ^ teilungen) und um das Volumen der einzelnen Fraktiot1 ^n quantifizieren. Auch Zelitriimmer und Agglomerate von ^ konnten quantitativ bestimmt werden. Tote Zellen wurden ihre Verschiebung im mittleren Zelldurchmesser erkannt. v ser-Diffraktometrie ist daher geeignet, die Qualitat einer le ^f lierung (z. B. durch Leberperfusion) zu best im men und ' ^ y ^ r
rlaubte dnderungen in der Zellpopulation wahrend der Suspension* ^ von Zellen zu verfolgen. Die Analyse von Humanblut erlau >
zwischen Thrombozyten und anderen Blutzellen zu UTltCirStv^n den. Die Populationen der Erythrozyten und der Gf a m tre,\- waren aufgrund ihres geringen Grofienunterschiedes nichi -lt .fJJ nen. Monozyten konnten wegen ihrer c.xtrcm klcinen ^ Blut nicht detektiert werden. Dies zeigt die Grenzen der \M
Key WOrdS'
Cell suspension • Laser diffractometry_ Pharm. Ind. 54. Nr. !!is,o«*
V O O Rudt ct al. - AnaKsis tn cell susf*
^Production
t o , C r o s c° P y is one m e t h o d to determine cell subpopulations and
a- * *U a n tt f y their fractions using cell size a n d morphological ranee. M i c r o s c o p i c e x a m i n a t i o n is relatively time-consu- r,i h ^0r many applications it can be replaced by particle sizing Iran- Wnen tne ce^ populatins are very different i n size. T h e
(Q 1 0 na l and very successful instrument is the C o u l t e r counter
^ u l t e r Electronics, K r e f e l d , F R G ) . T h e C o u l t e r counter is ba- rf?0_ ^e s*s l a n ce measurements, the height o f electric impulses
Fourier Lens
Scanning Receiver
i $ cU°resislance measurements, the height ot eiectnc unpui»c>
lj,e p l a t e d to the particle size. S i m i l a r instruments based on
% i , °U , l e r Pr i nc i p l e are on the market, some use a different
stem P r o c e s s i nS - T he CdI A n a l y s e r System Casey (Scharfe Sy-
for • ^e u l I mg e n , F R G ) calculates the area under the impulse t , p jS l 2 e determination. S i z i n g measurements o f cells have m u l -
de a pPn ca t i o n s . T h e y can even p r o v i d e i n f o r m a t i o n about cell
E 19 and r eP ! a c e staining methods (e.g. for epitheloid cell line
t^l UD- T h e cells s h r i n k o r deteriorate leading to a detec-
e decrease i n the mean diameter.
P l o y0 8 ^C ^a s t years *a s e r diffractometers are increasingly em- They * °r s'ze anaIysis as an alternative to the C o u l t e r counter.
an ,*are attractive because they are easier to handle and the k$$ h S *S Very rap^ (measurement a n d mathematical analysis new n ^ T he question however is to what extent can the
tr a (j .m e t n° d be e m p l o y e d to characterize cell populations, the
l jl v ^1 0 na l d o m a i n o f the C o u l t e r counter. To assess the suitabi-
l i ve r er diftractornetry we analysed h u m a n b l o o d and various
deh C C f I m*xtures differing i n quality (purity, dead cells, cell
n s- cell aggregates).
* Materials and methods
jj*' Materials
buffj C e J , s were obtained by perfusion o f rat liver using perfusion W$ u cnemica,s described i n detail elsewhere [2. 3]. B l o o d
ve n o b l ai n e d from h u m a n volunteers and analyzed directly after
l(w Us Puncture. A l l cell suspensions were d i l u t e d with physio-
a l salt solution for laser diffractometer analysis.
2'2- Methods
' * Isolation of liver cells (cell suspension I)
SP r a C e I ! s w e re obtained by perfusion o f the liver o f young male
bUft\pu*r~^a wley rats. After i n situ perfusion with a calcium-free a cj r * °r a p p r o x i m a t e l y 20 m i n . the liver was removed and in na$eSe^00P system perfused with a buffer c o n t a i n i n g collage-
U)e| a n c* c a l c i u m (20 m i n ) . T h e proteolytic enzyme dispersed
re i t l'V e r- l ne intact cells were suspended in buffer and filtered to
Spe Ve r e m a i n i n g crue pieces o f liver tissue [2, 3J. this cell su-
chv S l^n contained hepatoevtes (parenchymal cells), nonparen-
sUsn2 C e I , s <K uP f f e r cells, endothelial cell's), and cell debris (cell
for j ?*0.n T h e cell suspension I was d i v i d e d in equal parts celic / a t' ° n o f hepatoevtes (cf. 2.2.2.) a n d o f nonparenchvmal
l l s (cf. 2.2.3.)
^* • Eolation of hepatocytes (cell suspension II)
f
uJL
CeIIs from suspension I were washed three times by centri-djs 10n (2 m i n . 15 g). T h e nonparenchvmal cells remained i n pa/ /r s' ° n d u r i n g this centrifugation at l o w g number, the he-
^Itu es Were o r j t am e d as cell pellet and re-suspended i n cell
r na i [e m e d i u m (cell suspension II). T h i s suspension contained ann - hepatoevtes and c o n t a m i n a t i o n bv nonparenchvmal cells
n d cell debris [2. 3].
* • Isolation of nonparenchymal cells (cell suspension III)
t0 c° pl a m nonparenchymal cells the enzyme pronase was added
non S ^r°m susPens*on I- Pronase lyses hepatoevtes whereas the
^ ^a r e nc h y m a l cells are relatively resistent against this en-
pjj^* '^fter 1 h most o f the hepatoevtes were lysed and the non- g) -j.fjchvmal cells were obained by centrifugation (4 m i n , 400
Tn e e C e' l s localize in the pellet, the debris remain in dispersion.
s j0 n Pellet was resuspended i n cell culture m e d i u m (cell suspen- ds, " I [4]. T h e suspension contained m a i n l y Kupffer cells, en-
M( we'i a'c e , l s-and c o n t a m i n a t i o n by hepatoevtes and cell debris.
&an L° ^l n e hepatoevtes were dead ( > 99 %, determined bv Trv-
n b lu e staining).
\.
Ind 54-Nr- 'I (1992>
t al. — Analysis in ecl! suspensions
Fig. 1: Set up o f laser diffractometer. T h e laser light is diffracted from the suspended particles forming a Fraunhofer diffraction pattern on a multi-element detector. T h e form o f the pattern is size-spezific, its intensity correlates with the particle number.
2.2 A. Determination of size distribution by laser diffractometry T h e laser diffractometer is based on the diffraction o f laser light from spherical particles (Fraunhofer diffraction) [6—8]. T h e par- ticles are suspended i n a transparent l i q u i d (e.g. water, organic liquids), an extended laser beam passes through the sample cell and the diffracted laser light is detected by a multi-element ring detector ( F i g . 1). T h e diffraction pattern is size-specific, its i n - tensity is a function o f the particle number. T h e superposition of different size-specific patterns each v a r y i n g i n its intensity as a function o f the particle number can be mathematically resolved to calculate a v o l u m e d i s t r i b u t i o n [9, 10]. T h e theory is based on spheres. T h e method is therefore less suitable for rod-like or needle-shaped particles but can be employed for spherical cells.
Measurements were performed in physiological salt solution using the suspension cell o f the Sympatec laser diffractometer (Sympatec, Clausthal-Zellerfeld, F R G ) . C e l l suspension was added until the instrument indicated o p t i m u m measurement c o n d i t i o n s (diffraction intensity). F o r the measurement o f the liver cell suspensions a 50 m m lense was used corresponding to a measuring range o f 0.5 to 100 urn. H u m a n blood was measured using a 20 m m lense corresponding to a measuring range o f 0.1 to 74.5 urn.
3. Results and discussion
3.1. Characterization of cell suspension I (cf. 2.2A.) T w o cell preparations ( A , B) differing i n quality were investiga- ted. T h e size d i s t r i b u t i o n curve o f preparation A exhibits 3 peaks at approx. 3, 10. and 28.0 u m ( F i g . 2 A ) . T h e small peak repre- sents the cell debris, the peak at 10 u m the fraction o f nonparen- chymal cells which are reported to possess a size between approx.
8 and 11 u m . Hepatoevtes are in the range o f 25 to 35 u m leading
p a r t i c l e size [ p m ]
1 U p a r t i c l e size [ j i m ]
Fig. 2: V o l u m e distributions o f cell suspensions I obtained from liver perfusion (preparation A : left; preparation B : right; H - he- patoevtes, N P = nonparenchymal cells, D = debris).
Table 1: V o l u m e fractions o f cell p o p u l a t i o n s and c o n t a m i n a t i o n by debris i n cell suspension I isolated from rat liver.
P r e p a r a - t i o n
D e b r i s ( 0 . 4 5 - 4 . 5 u m )
N o n p a r e n - c h y m a l cells ( 4 . 5 - 1 5 . 0 um)
Hepatocytes ( 1 5 . 0 - 4 3 . 5 urn) A
B
6.8 % 0.9 %
1 7 . 0 % 1 1 . 0 %
76.2 % 88.1 %
to the peak at 28.0 u m . T h e curves are v o l u m e d i s t r i b u t i o n s w h i c h e x p l a i n the relatively large area under the peak o f the he- patocytes. T h e hepatocytes are 60 % i n number o f the l i v e r cells [11] but a p p r o x . 80 % i n v o l u m e . T h i s is well i n agreement with the 76 v o l u m e % o f the hepatocyte peak (Table 1), T h e peak o f the debris is relatively large i n preparation A . D u r i n g prepara- t i o n B less cells were destroyed d u r i n g the dispersion o f the liver.
T h e peak o f the debris is d i s t i n c t l y smaller ( F i g . 2B), the area d r o p p e d from 6.8 to 0.9 % (Table 1). Therefore laser diffracto- metry p r o v i d e i n f o r m a t i o n about the q u a l i t y o f the preparation.
Q u a l i t y being defined as percentage o f total cells i n contrast to debris. In a d d i t i o n , the fraction o f a s u b p o p u l a t i o n i n the total cell suspension can be determined (cf. 3.3.).
3.2. Characterization of cell suspension II (hepatocytes) A relatively large fraction o f debris and n o n p a r e n c h y m a i cells r e m a i n e d after centrifugation i n the cell suspension II. There are still distinct peaks for debris and n o n p a r e n c h y m a i cells ( F i g . 3 A ) . Centrifugation i m p r o v e d the purity o f the hepatocyte p o p u l a - t i o n . H o w e v e r , the isolation A was less successful than isolation B as i n d i c a t e d by the v o l u m e percentage o f hepatocytes (88.1 a n d 92.6 %, respectively, Table 2). T h i s is also reflected by the m i s s i n g debris peak and the relatively small n o n p a r e n c h y m a i peak i n preparation B ( F i g . 3B).
3.3. Characterization of cell suspension III
(nonparenchymai cells) ^ T h e v o l u m e d i s t r i b u t i o n o f cell suspension H I ( Pr ePa r a t l 0^tj n g
exhibits 3 large peaks, the largest peak at 23.5 u m repress hepatocytes ( F i g . 4 A ) . T h e peak o f n o n p a r e n c h y m a i cells l<*
at approx. 9 u m is relatively small i n d i c a t i n g no successfu P fication o f the cell p o p u l a t i o n . There is also a t h i r d fra . above 50 u m . T h i s fraction consists o f cell aggregates to ^ d u r i n g the p u r i f i c a t i o n treatment. These aggregates cou ^ identified as dead hepatocytes by Trypan blue staining. 7^.1^
o f the hepatocytes shifted from 28.0 to 23.5 u m . T h i s indi that the cells are dead w h i c h c o u l d also be c o n f i r m e d by - ^ blue staining. T h e cells s h r i n k or deteriorate leading to a ^ ^ j . in cell diameter. T h e separation o f cells from debris was r vely high as i n d i c a t e d by the s m a l l tail o f the v o l u m e d is t n~ . curve i n the range up to 4 u m (5.7 % debris. Table 3). The resulted from hepatocytes lysed by pronase treatment.
In contrast, the i s o l a t i o n o f n o n p a r e n c h y m a i cells i n Pr ej ?a v 0. B was more successful as i n d i c a t e d by the large peak of tn lume d i s t r i b u t i o n curve at 7 u m ( F i g . 4 B ) . T h e v o l u m e
increased from 21.7 to 45.5 % at simultaneous reduction o ^ hepatocyte fraction (from 56.9 to 23.0 % . Table 3).Howeverafe d separation o f cells from the debris was less successful cornp ^ to preparation A . T w i c e the fraction o f debris remained in cell suspension after centrifugation (Table 3). Further p u n t i o n o f the n o n p a r e n c h y m a i cells can be achieved by cell c U^ . Qf techniques (e.g. adhesion to culture dishes and washing 0 nonadherent cells a n d debris). P r e p a r a t i o n B is the best pr ration w i t h regard to the y i e l d o f n o n p a r e n c h y m a i cells.
3.4. Analysis of human blood
A n a l y s i s o f h u m a n b l o o d yielded a b i m o d a l v o l u m e d i s1^ "1! ? ( F i g . 5). A s m a l l peak at 3 u m represents the thrombocytes ( meter 2 - 4 u m . 150 0 0 0 - 4 0 0 000 per ul [5]). T h e large pc were the erythrocytes ( 6 - 8 u m [5]) i n c l u d i n g the other vl
l lft lftft 9.1 1 19 I M p a r t i c l e s i n [ | i m ] p a r t i c l e size [ y m ]
Fig. 3: V o l u m e d i s t r i b u t i o n s o f cell suspensions II o b t a i n e d after purificating cell suspensions I (preparation A : left: preparation B : right; H = hepatocytes, N P = n o n p a r e n c h y m a i cells, D =» de- bris).
1 ift p a r t i c l e size [ y m ] p a r t i c l e size { y m ]
Fig. 4: V o l u m e d i s t r i b u t i o n s o f cell suspensions III obtai
nedi r pronase treatment and centrifugation o f cell suspension ^ ^ 1 $ . aration A : left; preparation B : right; N P = nonparenchyrna H = hepatocytes. A = agglomerates. D = debris).
Table 2: V o l u m e fraction o f hepatocytes a n d c o n t a m i n a t i o n by debris before p u r i f i c a t i o n (cell suspension I) and after centrifu- gation treatment to isolate hepatocytes (cell suspension II).
Hepatocytes D e b r i s
Prepa-
ration before centri- fugation
after centri- fugation
before centri- fugation
after centri- fugation A
B
76.2 % 88.1 %
88.1 % 9 2 . 6 %
6 . 8 % 0 . 9 %
5 . 2 % 0 . 6 %
Table 3: V o l u m e fractions o f n o n p a r e n c h y m a i cells andne^ r
cytes a n d c o n t a m i n a t i o n by debris and agglomerates a ft e r. ^ iaK ase treatment o f cell suspension I and centrifugation to is°
n o n p a r e n c h y m a i cells (cell suspension III).
Prepa- ration
D e b r i s (0.45 to 4.5 \im)
N o n p a r e n - c h v m a l cells
"(4.5 to 15.0 u m )
Hepatocvtes (15.0 to 43.5 u m )
Aggl°"
merates 0 43.5M**
A B
5 . 7 % 1 3 . 4 %
2 1 . 7 % 4 5 . 5 %
56.9 % 2 3 . 0 %
1 5 . 7 * 1 8 ^ >
Pharm. Ind. 5 4 . Nr. M ilJfi Rudt et al. — Analysis in cell susfX0*^
5 1« 15 p a r t i c l e size [ y m ]
& V o l u m e d i s t r i b u t i o n o f h u m a n b l o o d analyzed by laser
^ " r a c t o m e t r y (small peak: thrombocytes ( T ) , large peak: ery- nrocytes, lymphocytes, a n d granulocytes).
s being in the range 8— 12 u m (granulocytes, lymphocytes [5]).
fra e c t e c* cel 's were below 12.2 u m . T h i s means that the low
cU o n o f larger s i z e d monocytes (14—17 u m ) was not detected.
e number o f 140—700 monocytes per uJ b l o o d is too l o w . S p a r e d w i t h the large n u m b e r o f erythrocytes a n d therefore
°w lh e detection l i m i t o f the laser difTractometer. A higher J /0'u t i o n ° f the d i s t r i b u t i o n between 6 a n d 12 u m cannot be
c inedDV laser diffractometer. F o r this a p p l i c a t i o n a C o u l t e r
un t e r appears more suitable (e.g. use o f a 30 o r even 15 u m H i l a r y ) .
Further applications
"Jajor advantage o f the m e t h o d is the use o f any dispersion
^ Qium w h i c h is transparent for light. Residues o f electrolytes
w- l ne cell isolation process o r cell culture m e d i u m interfere
n assays based on c o n d u c t i v i t y measurements (e.g. C o u l t e r
H iU?ler) - Agglomerates in the range of 50 u m are no p r o b l e m for
l!?e laser - - -~
L° u l t e r (
a$ 100
• debris
i h , ~~o • •
^e laser difTractometer but w i l l block the 50 u m capillary o f a
* counter. T h i s requires the use o f a larger capillary such - J u m , setting the lower detection l i m i t to 2 u m . A part o f
f h e debris cannot be detected anv more whereas the laser dif-
fer- ' . . _ _ C d l l l l U l U C U C l t A . I t v j a i i > u i u i \ .
^ l o m e t e r has a measuring range from 0.5 to 87.5 u m . T h e
U pP e r l i m i t for a C o u l t e r counter 100 u m capillary is about 80
A£ya n t a g e s o f the C o u l t e r counter are the d e t e r m i n a t i o n o f an
a bs o l u t e cell n u m b e r per v o l u m e unit a n d its sensitivity to detect
a fe* large cells i n a large p o p u l a t i o n o f smaller cells. A C o u l t e r
c°un t e r appears therefore more suitable for detecting the l o w
n u m b e r o f monocytes (14—17 j i m , 140—700 monocytes per u l blood) i n h u m a n b l o o d w i t h a d o m i n a t i n g number o f red b l o o d cells (6—8 u m , 4—6 m i l l i o n per u l , [5]). A n u m b e r d i s t r i b u t i o n can, however, also be obtained by a laser diffractometer using a defined v o l u m e o f cell suspension a n d c o n v e r t i n g the v o l u m e d i s t r i b u t i o n mathematically to a n u m b e r d i s t r i b u t i o n . However, the mathematical conversion is less precise than the direct meas- urement o f the n u m b e r d i s t r i b u t i o n ( C o u l t e r counter).
4. Conclusions
Laser diffractometry is a very r a p i d m e t h o d to determine the c o m p o s i t i o n o f m i x e d cell populations a n d possible c o n t a m i n a - tion by debris o r cell agglomerates. T h e v o l u m e d i s t r i b u t i o n s obtained were i n good agreement with literature data. In contrast to conductivity-based assays, salts from the isolation process or cell culture m e d i u m d o not interfere with the assay. Laser dif- fractometry is as a m u l t i p l e particle-counting technique, how- ever, less sensitive i n detecting a few large cells i n a cell suspen- sion c o n t a i n i n g a large n u m b e r o f small cells (e.g. monocytes i n h u m a n b l o o d with m a n y erythrocytes) o r vice versa.
5. References
[1] Scharfe System A p p l i c a t i o n Report, Scharfe System, R e u t l i n - gen, F R G (1990) - [2] Seglen, P. O . , Preparation o f Isolated R a t L i v e r Cells, M e t h o d s C e l l B i o l . 13, 29 (1976) - [3] Brauer, A . , Hepatocyten-Zellkulturen — in v i t r o Testsystem z u r O p t i m i e - rung neuer A r z n e i f o r m u l i e r u n g e n , P h . D . thesis, K i e l U n i v e r s i t y (1991) - [4] M u n t h e - K a a s , A . C , Berg, T., Seglen, P. O . , Seljeld, R., J . E x p . M e d . 141, 1 (1975) - [5] Wheater, P. R . , B u r k i t t , H . G . , D a n i e l s , V. G „ F u n k t i o n e l l e H i s t o l o g i c p. 24—37, U r b a n &
Schwarzenberg. M u n c h e n — W i e n — etc. (1979) — [6] Fraunhofer, J . , G i l b e r t s A n n a l . Phys. 56, 193 (1817) - [7] M u l l e r , B . W., L u c k s , J . S., Stampa, B . , M u l l e r , R . H . , P h a r m . I n d . 52, 789 (1990) - [8] A i r y . G . B . . Trans. C a m b . P h i l . Soc. 5, 283 (1835) - [9] P h i l i p s , D . L . , J . A C M 9, 84 (1962) - [10] T w o m e y , S., J . A C M 10,97 (1963) - [ 1 1 ] Bridges, J . W., T h e U s e o f Hepatocytes in Toxicological Investigations, i n : Testing for T o x i c i t y . J . W.
G a r r o d (ed.), p. 125—143, Taylor a n d F r a n c i s L t d . , L o n d o n (1981)
For the authors: Prof. D r . R . H . M u l l e r , Pharmazeutisches Institut der U n i v e r s i t a t K i e l , Gutenbergstr. 76—78, W - 2 3 0 0 K i e l ( F e d . R e p . o f G e r m a n y )
^ ei a|. — Analysis in cell suspensions
