A Method for Isolating Large Numbers of Viable Disaggregated Cells from Various Human
Tissues for Cell Culture Establishment
Author(s): Ruth E. Gibson-D'Ambrosio, Mervyn Samuel and Steven M. D'Ambrosio
Source: In Vitro Cellular & Developmental Biology, Vol. 22, No. 9 (Sep., 1986), pp. 529-534
Published by: Society for In Vitro Biology
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IN VITRO CELLULAR & DEVELOPMENTALBIOLOGY
Volume 22, Number 9, September1986
? 1986 Tissue Culture Association,Inc.
A METHOD FOR ISOLATINGLARGE NUMBERS OF VIABLE DISAGGREGATEDCELLS
FROM VARIOUS HUMANTISSUES FOR CELLCULTURE ESTABLISHMENT
RUTH E. GIBSON-D'AMBROSIO,MERVYNSAMUEL, ANDSTEVEN M. D'AMBROSIO
Departmentsof Radiologyand OB-Gyn,The OhioState University,Columbus,Ohio 43210
(Received8 November1985;accepted11 March1986)
SUMMARY
A method is described for the isolation of large numbers of viable disaggregated cells from human
tissues. This method combined the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml
trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid
(EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney, liver, lung, and skin were
separately minced into approximately 1-mm3 pieces. The pieces were placed in a sterile bag containing
60 ml of calcium- magnesium-free phosphate buffered saline, the appropriate enzyme (0.1 mg/ml
trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then
placed into the blender and mixed at a low speed for 3 to 20 min at room temperature. After a single cell
suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added to the cell
suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts
solution containing 5% bovine calf serum was added and the entire cell suspension passed through a
tissue sieve (100 mesh, 140 pm) and the cells collected by centrifugation. These cells were then
resuspended into the appropriate culture medium. In comparison to other methods for establishment of
cell cultures from human tissues, the method described requires shorter incubation times with
relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of
cells per tissue with 86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells
attached to the culture vessel within 24 h. Variation of the time and concentration of digestive enzymes
can be used to select different cell types for culture.
Key words: primary cell culture; human culture establishment; tissue disassociation.
concentrations of single or multiple proteolytic enzymes
at 40 to 370 C. Physical stirring or vigorous agitation are
often used to aid the disruption of the tissue matrix. Often
the procedures used to isolate and then culture human
cells result either in a high number of cells with low
viability or a low yield of cells with high viability. A
number of exogenous factors, such as enzymes, temperature, dissociation medium, osmolarity, pH, and time the
tissue is exposed to the dissociation solution, seems to be
important for establishing viable cell culture from
mammalian tissues (14). To obtain a large number of
viable, functionally active cells for use in the establishment of large-scale primary cultures, we describe a
method that uses short (3 to 20 min) incubation times and
low concentrations (0.1 mg/ml trypsin or 0.5 mg/ml
collagenase or both) of proteolytic enzymes in conjunction with the mechanical action of a Stomacher lab
blender.
INTRODUCTION
Studies using human tissues and cells can probably
provide more relevant mechanistic information on human
disease processes and biochemical and metabolic functions than cells derived from other species. Cells derived
from the internal organs may further help define cellular
and organ-specific
responses. The development of
culture systems for cells derived from many different
human organs can provide models for studying toxicity,
mutagenesis, and transformation. Some of the major
limitations to the development of actively replicating cell
cultures from human organs include availability of viable
tissue, methods for obtaining large number of viable
cells, maintaining the cells in long-term culture, and
overgrowth of the cell cultures with fibroblasts. To help
overcome some of these limitations, we developed
procedures for isolating large numbers of viable cells
from a variety of human organs.
Many methods have been described (5,6,13) using
various combinations
of physical, enzymatic,
and
chelating agents to isolate single, viable cells from human
tissue samples for the establishment of cell cultures.
Several reviews (1,9,15) discuss both the positive and
negative aspects of many of these methods. Many of these
procedures use incubation times of 1 to 12 h, with various
MATERIALSAND METHODS
Tissue. Human fetal intestine, kidney, liver, lung, and
skin tissue was obtained from the Ohio State University
Hospital immediately after suction curettage. The tissue
(whole organ) was then placed immediately into ice cold
Hanks' balanced salt solution (4), pH 7.2 (H-BSS),
529
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GIBSON-D'AMBROSIOET AL.
530
ROOM
TISSUE OBTAINEDFROMHOSPITALOPERATING
I
WASHTISSUE IN PBS CONTAININGANTIBIOTICS
I
DISECT DESIREDSECTIONFROMORGAN
CUT TISSUE INTO 1 to 2 mm3PIECES
BAG
INTO STOMACHER
PLACETISSUE FRAGMENTS
CONTAINING60 ml TRYPSIN(0.1mg/ml) + EGTA (0.1 mM)
OR COLLAGENASE
(0.5mg/ml) + EGTA (0.1 mM)
I
LABBLENDERWITH
PLACESEALEDBAG INTO STOMACHER
PADDLESPEEDSET AT 150 TO 160 STROKESPER MINUTE
I
INCUBATE3 TO 20 MINUTESAT ROOMTEMPERATURE
I
ADD 10 ml CALFSERUMAND 130 ml COLDHANKS-BSS
I
PASS THROUGHTISSUE SIEVE (100 MESH,140 u)
I
COLLECTCELLSBY CENTRIFUGATION
TURE
IN
A
CULTURE IN APPROPRIATE MEDIUM
FIG. 1. Experimental flow chart for the isolation and culture
of viablecellsfromhumantissues.
consisting of CaCl2 (0.95 mM), KCI (5.3 mJM), KH2PO4
(0.44 mM), MgCl2 (0.49 mM), MgSO4 (0.40 mM), NaCI (137
mM), NaHCO3 (4.1 mM), Na2PHO4 (3.3 mM), D-glucose
(5.5 m.M), phenol red (0.03 mM), streptomycin (200
and Fungizone (500
ng/ml),
penicillin (200 U/ml),
ng/ml). The tissue was processed for cell culture within 2
h of surgery.
Tissue fragmentation and cell isolation. Described
below and outlined in Fig. 1 are the steps used in the
isolation of viable disaggregated cells from human
intestine, kidney, liver, lung, and skin. The tissues were
washed three times in a calcium- and magnesium-free
phosphate buffered salt solution (PBS) consisting of NaCI
(137 mM), Na2HPO4 (8.1 mM), KCI (2.6 mM)KH2PO4 (1.4
mM), and the above antibiotics, pH 7.2, to remove the blood
cells. The desired organ component was carefully dissected at
room temperature. Approximately 0.5 g or 2 cm3 of this tissue
was then placed onto a glass cutting block with approximately
0.5 ml of the proteolytic enzyme solution to help prevent the
tissue from attaching to the course cutting block. The cutting
block was a 40 X 75 X 5 mm Pyrex glass block that had been
chemically etched to form a rough working surface. The
corners of the blocks were cut off to allow its placement into a
100 mm diameter Pyrex glass petri dish. The tissue was then
minced into 1- to 2-mm2 pieces by slicing in opposite directions, using two sterile scalpel blades. The tissue fragments
from approximately 0.5 g or 2 cm3 of tissue were placed into
the sterile Stomacher bag containing 60 ml of the appropriate
digestive solution.
Cell dispersing solution. The PBS containing phenol
red (0.03 mM),
[ethylene bis(oxyethylenenitrolo)]tetraacetic acid (EGTA) (0.1 mM), and 0.1 % methylcellulose (low substitution with a viscosity of 350 to 550
centipoise, pH 7.8, was used. All enzymatic digestion
were carried out in this solution at room temperature.
Collagenase and trypsin were purchased from Worthington Biological, Freehold, NJ. The trypsin was 2X
crystallized and used at a 0.1 mg/ml concentration. The
collagenase was Worthington's type II and used at a
concentration of 0.5 mg/ml. EGTA was obtained from
Sigma Chemical Co., St. Louis, MO, and used at a final
concentration of 0.1 mM. Trypsin-EGTA was used with
intestine, kidney, lung, and skin; collagenase-EGTA was
used with liver.
The Stomacher lab blender was purchased through
Tekmar Co. (Cincinnatti, OH). It was the Model 80
connected to a variable voltage transformer. This voltage
regulator was set at approximately 90 V AC which reduced
the paddle speed from 380 to 430 strokes to 150 to 160
strokes/min. Once the tissue was placed in the bag, the
top closures were carefully folded down approximately 4
cm from the top. The bag was then placed into the
blender such that the blender's door was securely closed
with the bag's folded closure located immediately above
the door. At this time the voltage regulator was turned on
for 3 to 20 min depending on the digestive solution,
tissue, and extent of digestion desired.
Inactivation of proteolytic enzymes. After incubation,
10 ml of bovine calf serum (BCS) was immediately added
to the bag containing the digestive solution and cells.
After mixing by pipetting, 130 ml of 40 C H-BSS
containing 5% BCS was added to the 70 ml of cell
suspension. At this point the cell suspension was pipetted
TABLE 1
YIELD AND VIABILITYOF CELL
FROMHUMAN TISSUES
Tissue
Intestine
Kidney
Liver
Lung
Skin
Tissue Weight"
(g)
0.37 ?
0.18 ?
1.53 ?
0.58 ?
0.37 ?
0.13b
0.08
1.15
0.23
0.11
"Wetweight.
bMean? SD of 6 to 10 samples.
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Cell Yield
10 6 Cells/g Tissue)
42.8 ?
92.2 ?
144.0 ?
84.4 ?
43.7 ?
3.9
4.2
9.4
4.8
5.4
Viable Cells
(%)
89.6
86.4 ?
91.2 ?
92.1 ?
88.7 ?
4.4
5.2
3.4
5.2
7.1
531
ISOLATIONOF HUMAN CELLS
through a stainless steel sieve (100 mesh), with a pore size
of 140 am, to remove tissue fragments.
Cell culturing. After sieving, the cells were collected by
centrifugation at 160 X g for 5 min at 40 C. After pelleting
the cells were resuspended into the respective growth
media and counted for viability using trypan blue dye
exclusion. Cells were seeded for cell culture at a density
of 1 X 104 cells/cm' or placed over Percoll step
gradients for further purification.
RESULTS
Effect of proteolytic enzymes on cell yield. When the
tissue was cut into approximately 1- to 2-mm' pieces the
surface area for the proteolytic enzymes to act on was
greatly increased. This was best achieved by cross cutting
the tissue placed on top of a glass cutting block using two
scalpel blades (1). Various concentrations of trypsin (0.1
to 5.0 mg/ml) and collagenase (25 to 0.1 mg/ml) were
tested in the Stomacher lab blender for both cell yield
and viability of cells isolated from human intestine,
kidney, liver, lung, and skin. Trypsin was used with the
intestine, kidney, lung, and skin, and collagenase was
used with liver. A 2.5 mg/ml concentration of trypsin
destroyed most of the cells isolated from the tissues. A 1.0
mg/ml concentration of trypsin yielded a large number of
cells, but with low (20 to 30%) viability. Lower, i. e. 0.1
mg/ml, concentrations of trypsin greatly increased cell
viability (60%) but yielded relatively small number of
cells per tissue mass. The yield of cells was greatly
increased by the addition of 0.1 mM EGTA. In all of the
tissues tested 0.1 mg/ml trypsin plus 0.1 mM EGTA
yielded 108 to 10' cells/gm tissue with 87 to 92% viability
(Table 1). High concentrations (>1.0 mg/ml) of collagenase destroyed most of the liver cells. Lower concentrations (0.5 mg/ml) of collagenase increased cell yield and
viability. EGTA (0.1 mM) was found to be a necessary
addition to obtain a high cell yield and 92% viability
(Table 1). EGTA alone resulted in a low cell yield, poor
viability, and cell clumping.
Paddle speed. The paddle speed on the blender as
obtained from the manufacturer was 380 to 430
strokes/min. At that rate, the tissue was completely
dispersed yielding mostly subcellular particles. Very few
if any whole viable cells were observed under phase
contrast microscopy. To overcome this problem, we
attached a variable voltage transformer to the blender.
Setting the voltage regulator to 90 V AC, yielded 150 to 160
strokes/min. This paddle speed yielded the largest
number of viable cells per tissue.
Time of incubation. The time the tissue was incubated
with the proteolytic enzyme in the blender was found to
be critical for cell yield. Time of incubation was also
dependent on the tissue. Table 2 indicates our optimal
incubation times for each tissue tested. Kidney and
intestine required 8 to 10 min to yield viable single cells,
whereas lung, liver, and skin required 15, 15, and 20 min,
respectively. Using these incubation times and the paddle
speed and enzyme solutions described above, a suspension of single cells was obtained (Fig. 2 A). Very few
aggregated cells were observed. Lesser incubation times
yielded incomplete dissociation, whereas longer times
decreased the number of viable cells. The extent of tissue
disruption can be monitored by direct visualization in the
blender bag using phase contrast microscopy, and the
extent of tissue disruption can be controlled and used to
isolate cell types by varying the incubation time.
Cell culture. Cells isolated using the blender were
passed through a 140 Mm, 100 mesh stainless steel sieve.
This removed the remaining tissue fragments and yielded
a cell suspension containing mostly single cells (Fig. 2 B).
The cells were collected after centrifugation at 160 X g
for 5 min and then resuspended in their respective growth
medium. The cells were observed to attach to the tissue
culture plates within 24 h (Fig. 3). At a seeding density of
1 X 104 cells/cm2, approximately 40 to 55% were found
to attach to the surface of the dish within this time period.
The cell cultures were replicating actively, and the time
for primary culture to reach confluency was 7 to 10 d. The
characteristics of these cells in culture are described
elsewhere (2,3).
Comparison to other methods. A number of procedures
for establishment of cells in culture were compared
(Table 3). A major problem with the explant and
stirring/shaking water bath procedures was the overgrowth of the culture with fibroblasts. The extent of
fibroblast outgrowth in the explant culture could be
controlled by fragmentation in the culture dish, cell
cloning, and composition of culture medium. However,
the yield of epithelial cells was very low. Inasmuch as no
proteolytic enzymes were used in the explant procedure,
little if any damage was done to the parenchymal cells.
The stirring water bath procedure (7,8) requires that
tissue fragments be incubated for a long time with
relatively high concentrations of proteolytic or chelating
agents or both. We found that the cell yield was very low
and that parenchymal cells were destroyed by the
proteolytic enzymes. The cell dispersion method using
vigorous pipetting to release cells from a tissue matrix
used much lower concentrations of proteolytic enzymes
and thus increased the yield of parenchymal cells. Cell
viability was between 48 and 65%. However, cell yield was
very low. In all three of these procedures 14 to 24 d were
required for the culture to become confluent.
DISCUSSION
In this study, the mechanical action of a Stomacher lab
blender has been combined with low concentrations of
TABLE2
TIME REQUIREDFORISOLATIONOF SINGLE CELLS
Tissue
Intestine
Kidney
Liver
Lung
Skin
Time"
(min)
10
8
15
15
20
'Paddlespeedwas 150to 160strokes/min;temperaturewas 250 C.
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532
GIBSON-D'AMBROSIO ET AL.
FIG. 2. Cell suspension obtained immediately after dissociation of human fetal kidney using the Stomacher lab
blender. Cells before (A) and after (B) passing through a 140 pm, 100-mesh sieve. Notice that very few cell clumps
appeared after passing through the sieve. Kidney tissue was incubated for 10 min at room temperature with 0.1 mg/ml
trypsin and 0.1 mM EGTA. X 138.
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ISOLATIONOF HUMAN CELLS
533
FIG. 3. Phase contrast photomicrographof human fetal kidney cells in culture. Cells were isolated from fetal
kidney tissue using the blender, 0.1 mg/ml trypsin, and 1 mM, EGTA. Note the tightly packed cuboidal-shapedcells
andclonalgrowth.Cultureswere24-h-oldin an a MEM containing10%fetal calf serum.X 110.
proteolytic enzymes and EGTA to yield large numbers of
viable cells from various human tissues. Other methods
(13) using mechanical disruption of tissues to isolate cells
are based on cutting or stirring or both. They usually
result in a low yield of viable cells. The blender's two
paddles, unlike other mechanical devices, alternatively
compress the tissue sample in rapid succession. A
reduction of the paddle speed to 150 strokes/min was
necessary to yield the maximum number of viable cells.
With the mechanical agitation provided by the blender,
the concentrations of trypsin or collagenase required to
release cells from the tissue matrix were greatly reduced
from that described in other procedures (7). We also
found that the addition of EGTA was necessary to
optimize the yield of viable cells. Chelating agents like
EDTA and EGTA have been useful in the isolation of
cells in culture from the surface of the cell culture disk
(11) and in cells from tissue (12). We found that EGTA
greatly aided in the release of cells from tissue processed
in the blender. With the addition of 0.1 mM EGTA to the
proteolytic enzyme solution, the cell yield and viability
increased two- to threefold and cell clumping was
reduced.
The release of cells from tissue samples usually
requires incubation with trypsin or collagenase. Incubation with these proteolytic enzymes can damage the cell
surface which may be critical for cell culture and cell
function studies. Most procedures, depending on the
tissue, require incubation times between 1 and 12 h with
these proteolytic enzymes. The blender procedure also
requires incubation of the tissue with trypsin or
collagenase. However the amount required was reduced
to 0.1 and 0.5 mg/ml, respectively, and the incubation
time was as short as 3 min and no more than 20 min.
We determined conditions for isolating viable cells for
cell culture from human fetal intestine, kidney, liver,
lung, skin, and neonatal skin. The exact procedure
utilized for each tissue in terms of type and amount of
proteolytic enzyme and time of incubation was dependent
on the tissue. Soft tissues such as fetal kidney and
intestine required short incubation times with lower
amounts of proteolytic enzymes than did hard tissues
such as skin. The conditions for isolating cells from other
tissue may vary from those conditions described here for
our six human fetal tissues. We and others have used
slight modifications of this procedure to isolate cells for
culture from young adult rat mammary gland tissue (10),
neonatal and adult skin, adult canine liver hepatocytes
(Gibson-D'Ambrosio, unpublished), and adult human
gastric G cells (W. Gower, in preparation). Thus, the
Stomacher lab blender in combination with proteolytic
enzymes and EGTA should provide powerful approaches
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534
GIBSON-D'AMBROSIO ET AL.
REFERENCES
TABLE 3
COMPARISON OF METHODS FOR CELL DISPERSION
Percent of
Viability
Method
Primary explant
Stirring water
bath
NA
Comments
Fibroblast overgrowth with extended time
in culture
Culture establishment time (18-24 d)
Multilayering of cells, little monolayering
No exposure to proteolytic enzymes or
chelating agent
Intact tissue pieces
30 to 45 Culture establishment time (14-18 d)
Low cell yield
Exposed to proteolytic enzymes for 1 to
12h
Requires 2.5 mg/ml proteolytic enzymes
Parenchymal cells destroyed
Fibroblasts predominate
Vigorous
pipetting
Stomacher
Laboratory
blender
48 to 65 Incomplete dissociation
Uses 0.1 mg/ml proteolytic enzymes
Low cell yield
Little fibroblast contamination
Culture establishment time (up to 14 d)
85 to 93 Culture establishment time (7-10 d)
Requires short incubation times (8-20
min) with low concentrations (0.1 mg/
ml) of proteolytic enzymes
Control of paddle speed and time critical
for viable cell isolation
Yield large number of functional
parenchymal cells
Cells not aggregated
toward the isolation of viable cells from a variety of
humanand mammaliantissuesfor use in cell culture.
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Human fetal kidney cells in culture exhibit reduced levels of
DNA repair compared to human fetal dermal fibroblasts. In
Vitro 17:299-300; 1982.
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This work was supported by research grants from the National Institute of Environmental Health
Sciences, Bethesda, MD (ES3101) and the United States Environmental Protection Agency, Washington,
D. C. (R810146). We thank Gail Kimberlain for typing the manuscript.
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