1062-3604/04/3502- © 2004
Russian Journal of Developmental Biology, Vol. 35, No. 2, 2004, pp. 76–79. Translated from Ontogenez, Vol. 35, No. 2, 2004, pp. 105–109.
Original Russian Text Copyright © 2004 by Rogovaya, Vasiliev, Kiselev, Terskikh.
Living tissue equivalents are widely used for repair
of organ and tissue defects. Collagen gel is mostly used
as a biomatrix in living tissue equivalents, which repre-
sents the most physiological substrate allowing simula-
tion of the histotypical growth of ﬁbroblasts. Fibro-
blasts contract the collagen gel and produce a structure
resembling connective tissue, which was called “living
derma equivalent” (Bell
, 1979). Later, a living
skin equivalent was developed (Bell
which was based on collagen gel populated by ﬁbro-
blasts and with keratinocytes grown on its surface. Such
a skin equivalent was successfully used for closure of
wounds in rats and then in clinical practice (Bell and
Cultures of human ﬁbroblasts were used for treating
, 1994), granulating wounds and
, 1998), and urogenital ﬁstulae
Collagen gel is usually populated by a suspension of
ﬁbroblasts but ﬁbroblasts grown on microcarriers can
also be used. In this form, ﬁbroblasts are easily trans-
ported and stored in cryobanks.
The behavior of human ﬁbroblasts embedded in col-
lagen gel was extensively studied (Rudneva
1990). The present work was aimed at studying the
properties of a connective tissue equivalent produced
by embedding the ﬁbroblasts grown on microcarriers in
MATERIALS AND METHODS
Isolation of collagen.
Type I collagen was obtained
from tail tendons of laboratory rats weighing 200 g.
The amputated tails were immersed in 70% ethanol for
1–2 h. All subsequent manipulations were performed
under sterile conditions. Tendons were isolated suing
forceps and scissors and placed in 0.001% acetic acid.
Tendons from 10 tails were used to prepare one liter of
Collagen was extracted at 4
C for 48 h and the solu-
tion was then centrifuged at 2500 rpm (4
C, 2 h). The
supernatant was poured off in a sterile vessel and stored
C. The mass proportion of collagen in the obtained
solution was determined by drying to a constant weight.
Preparation of collagen gel.
Sterile 0.34 M NaOH
was mixed with concentrated (
10) nutrient medium
199 at 1 : 2 and 10% embryonic calf serum (Biolot,
St. Petersburg) and 9 ml 7.5% sodium bicarbonate were
added per 100 ml mixture. The obtained mixture was
combined with a cooled collagen solution in acetic acid
at 1 : 4 and placed on ice to prevent fast gelatinization.
At this stage a suspension of ﬁbroblasts or ﬁbroblasts
on microcarriers was introduced in the mixture and it
was poured off in 35 mm Petri dishes (Nunc, Denmark)
with nonadhesive surface.
Preparation of microcarriers.
cytopol (Biolot), 200–300
m in diameter, were
washed three times by Hanks solution, ﬂooded by
Eagle medium, and left for swelling for two–three days.
The microcarriers thus prepared were sterilized by
autoclaving and stored in Eagle medium in a refrigera-
tor for a year. For better cell attachment, the microcar-
riers were, before use, additionally treated with
0.1 mg/ml collagen (37
C, 20 min) and washed by
Hanks solution two times and by Eagle medium once.
Isolation and cultivation of ﬁbroblasts.
human ﬁbroblasts were obtained from three to ﬁve-
week abortive material. The embryo was washed sev-
CELL DIFFERENTIATION AND PROLIFERATION
Use of Human Fibroblasts Grown on Microcarriers
for Formation of Connective Tissue Equivalent
O. S. Rogovaya, A. V. Vasiliev, I. V. Kiselev, and V. V. Terskikh
Kol’tsov Institute of Developmental Biology, Russian Academy of Sciences, ul. Vavilova 26, Moscow, 119991 Russia
Received December 19, 2002; in ﬁnal form, September 19, 2003
—Living equivalents of tissues, speciﬁcally those produced on the basis of ﬁbroblasts and collagen
gel, are widely used for repair of organ and tissues defects. In clinical practice, it is more convenient to use the
ﬁbroblasts grown on microcarriers or such a connective tissue equivalent when the ﬁbroblasts on microcarriers
are embedded in collagen gel. We studied the properties of a connective tissue equivalent produced by embed-
ding the ﬁbroblasts grown on microcarriers in collagen gel for its prospective use in clinical practice. According
to our results, the optimal time of use of the living tissue equivalent amounts to three–four days after embedding
of ﬁbroblasts on microcarriers in gel. At that time, contraction only begins, which facilitates manipulations with
: ﬁbroblasts, microcarriers, collagen gel, living tissue equivalent.