(03401) Chun SONG et al.:Experimental Study of Rat Beta Islet Cells Cultured under Simulated

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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):47-50 CN 31-1300/Q

Experimental Study of
Rat Beta Islet Cells Cultured under Simulated Microgravity Conditions
Chun SONG1,2*, Xiu-Qing DUAN2, Xi LI3, Li-Ou HAN2, Ping XU2, Chun-Fang SONG2 and Lian-Hong JIN4
( 1Key Laboratory of Cell
Transplantation of Ministry of Health, First Affiliated Hospital of Harbin
Medical University, Harbin 150001, China;
2Department
of General Surgery, First Affiliated Hospital of Harbin Medical University,
Harbin 150001, China;
3National
Key Laboratory of Harbin Veterinary Research Institute of Chinese Agriculture
Academy, Harbin 150001, China;
4Tissue
Engineering Research Center, Histology and Embryology Staff Room, Harbin
Medical University, Harbin 150086, China )

Abstract To observe the effects of simulated microgravity on beta
islet cell culture, the survival rates and the insulin levels of the isolated
rat islet cells cultured at the micro- and normal gravity conditions were
compared. The survival rates of the cells cultured were determined by acridine
orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of
the cells was observed by electromicroscopy. Insulin levels were measured by
radioimmune assays. Our results show that the cell number cultured under the
microgravity condition is significantly higher than that under the routine
condition (P<0.01). Some tubular structure, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7 after the cultivation shown by transmission electromicroscopy. There were also abundant secretion particles and mitochondria in the cytoplasma of the cells. Scanning electromicroscopy showed there were holes formed between each islets, possibly the connecting points with the nutrients transport tubules. The microgravity cultured group also has the higher insulin levels in the media when compared with the control group (P< 0.01). Our results indicate that microgravity cultivation of islet cells has advantages over the routine culture methods.

Key words simulated microgravity; islet cell; electron
microscopy; radioimmunoassay

Clinical and experimental evidence has shown that
adequate islet cells transplantation is an ideal approach to treat
insulin-dependent diabetes mellitus. How to make islet cells three-dimensional
growth and differentiation, and become functional tissue in vitro, has
been a hot spot in investigations. The islet cell culture under microgravity
condition was studied here by using a rotary cell culture system (RCCS). The
survival rate, morphology and secretory function of the islet cells cultured in
this condition have been observed.

Materials and methods

Animals and reagents

Male Wistar rats, 250–300 g, were obtained from the
Experiment animal laboratory, First Affiliated Hospital of Harbin Medical
University. Hank’s solution and collagenase V were obtained from Gibco (USA),
and acridine orange (AO) and propidium iodide (PI) from Sigma (USA). RPMI-1640
medium and fetal calf serum were purchased from Hyclon (USA). Ficool-400
purified solution and dithizone (DTZ) were purchased from Sigma (America).
Insulin radio-immunity kit was obtained from Isotope graduate school, Chinese
Atomic Science Institute for research.

Experimental groups

Wistar rats were random divided into three groups. Group
I: control group, fresh islets (n=8); group II: flask-cultured (n=8);
group III: bioreactor-cultured (n=8). Each group was subjected to the
cultivation under normal gravity and the microgravity (RCCS, Synthecon, USA)
conditions.

Collagenase V preparation

5 mg collagenase V was dissolved in 2 ml Ca2+/Mg2+-free Hanks’
balanced salt solution supplemented with 100 u/ml penicillin and 100 mg/L
streptomycin.

Islet isolation and purification

Pancreas was exposed by aseptic operation and injected
with collagenase V on the multiple spot. Total pancreas was excised after
distended. Capsule was removed with scissors. Chopped pancreas tissues were
collected in gradient centrifuge tube with collagenase V and blew and beat
gently with pipette. The digest was shifted in the constant temperature incubator
at 37 ℃ (5% CO2), agitated
or blew and beat one time every 5 min and stopped with Hank’s solution (10%
calf serum) after 30 min and passed through a 600-mm mesh and then purified
using Ficool-400 density gradients after centrifuged. 250 g/L 4 ml, 230 g/L 2
ml, 200 g/L 2 ml and 110 g/L 2 ml Ficool-400 purified solution were added one
by one. 2 ml Hank’s solution was finally added. Islet cells from every two
interfaces besides 110 g/ L and Hank’s solution interfaces were collected after
centrifuged and washed two times in Hank’s solution. Pancreas was separated
into 50–350 mm many islet cell masses. The final cell pellet was resuspended in
RPMI-1640 culture medium with 20% fetal calf serum, 100 u/ml penicillin and 100
mg/L streptomycin, placed in culture flasks or RCCS besides fresh islets and
incubated at 37 ℃(5% CO2 and 95% air).

Islet identification

Islets were specifically stained by DTZ. 10 mg DTZ was
dissolved in absolute ethyl alcohol (50 ml concentrated NH4OH ),
supplemented with 12 ml Hank’s solution. Just before using, the preceding
solution was diluted with Hank’s solution (pH 7.8) by 1 to 100, passed through
a 0.22 mm filter membrane. Islet suspension was mixed with DTZ and placed 10
min and identified by light microscope.

Islet viability assessment and survival rate

Stock solution (AO: 670 mmol/L, PI: 750 mmol/L) was
prepared with Dulbeccos solution(isotonic phosphate buffer solution) and kept
in the dark place at 4 ℃. Just before
using, 0.01 ml AO and 1.0 ml PI were mixed, diluted by ten times with Dulbeccos
solution, passed through a 0.22 mm filter membrane, mixed 10 min with prepared
islet and observed and taken photograph by fluorescence microscope. Green (AO)
and red (PI) fluorescence were simultaneously seen at 510 nm grating light filter.
Islet survival rate cultured for days 3, 7 and 14 in stationary flaskes or
microgravity bioreactors was measured by AOPI double-staining. Islet survival
rate was showed with the percentage that live cells numbers were live cells and
died cells total amount. Each sample was repeatedly calculated four times, take
its average value [1].

Insulin level of culture fluids

Day 3, 7, 14, 21 and 30 islet cells culture fluids from
group II and group III were measured by radioimmunoassay.

Electron microscopy

Islet cells suspension cultured for 7 days from group II
or group III and from group I were respectively centrifuged and slowly added 3%
glutaraldehyde and observed by transmission electron microscopy (TEM) and
scanning electron microscopy (SEM).

Statistical analysis

Differences between islet survival rate were analyzed by
two sample rate u test. Unpaired Student’s t test was used to
analyze the differences between insulin level. P<0.05 was considered to be statistically significant.

Results

Survival rates of islets

Islet survival rate in stationary flasks or microgravity
bioreactors was measured by AO-PI double-staining. The result showed that there
was significant difference between bioreactor-cultured and flask-cultured on
day 7 and 14 (P<0.01). There were more cells survived in the Bioreactor cultivation group (Table 1).

Table 1 The different time survival rate of islet in
groupII and groupIII

^*P<0.01 vs groupⅡ.

Insulin
levels in the culture fluids

Insulin level of day 3, 7, 14, 21 and 30 islet cells
culture fluids from group Ⅱ and group Ⅲ revealed that bioreactor-cultured were higher than
flask-cultured and bioreactor-cultured functioned better (Table 2).

Table 2 Insulin level of culture
fluids

^*P<0.01 vs groupⅡ. Data are represented as x±s

Islet morphology observation

The islet morphology observation are shown in Fig. 1–Fig.
6.

Fig. 1 Fresh islet cells (TEM ×5000)

α 、β and δ cells were observed, which
presented high electron density secretory granules and well-formed mitochondria
and rough surfaced endoplasmic reticula.

Fig. 2 Fresh islet cells (SEM ×200)

Islet cells roughly aggregated into insulae,formed cells cluster and were
loosely connected.

Fig. 3 Day 7 flask-cultured cells (TEM ×8000)

Islet cells were closely connected, vacuolar degeneration in mitochondria,
lesser secretory having granules.

Fig. 4 Day 7 flask-cultured cells (SEM ×1500)

Countless small islets formed islet cells cluster, Islets arranged
intensively, no cleft and indistinct boundary.

Fig. 5 Day 7 bioreactor-cultured cells (TEM ×6000)

Islet cells presented well-formed secretory granules and mitochondria.
There were long and wide channel-like structures between arrays of islet cells
(namely nutritional channels).

Fig. 6 Day 7 bioreactor-cultured cells (SEM ×10000)

External surface of islet cells had cleft and cavity-like areas, which
probably represent the inlets into nutritional channels.

 

Discussion

In recent years, research in the field of islet
transplantation has made breakthroughs in some aspects of this treatment.
However the problem of not getting adequate islet cells for transplantation has
become a serious obstacle in the treatment. As an attempt we have cultured rat
islet cells in microgravity condition, and have observed the survival rates,
morphology and the insulin secretory function of the cells.

Developed in the space biological research projects,
microgravity tissue engineering technique has already been used in cultured
cells in vitro, and its technical goal is to establish a
three-dimensional culture system of animal cells [2].

The RCCS employed in the present research forms a model
simulating the microgravity condition. The cells were rotated together with the
vessels in the RCCS, forming a homogeneous fluid orbit in the media, simulating
the great mass of the gravity effects. The cells in RCCS can breath in O2, and out of
CO2 through
membrane fusion and gas exchange, designed to remove air bubbles from the
culture vessel and prevent turbulence, which may affect cell growth. The
cultured islet cells in RCCS had a better morphology and higher survival rate
after 7 days’ culture. This indicates that culture in microgravity provides
better growth environment for the islet cells. The insulin levels in the
bioreactor-cultured cells media under the microgravity condition was higher
when compared with flask-cultured islets, suggesting an increased islets cell
secretory function, and a better survival rates. This may have the potential in
the preperation of the cells for transplantation.

On the 7th day cultured under simulated microgravity
conditions electron microscopy revealed there were many nutritional channels
between arrays of islet cells containing electron dense secretory granules, and
there were also well-developed mitochondria, resembling the ultrastrucures of
live islets cell. Fig. 6 shows that there are many small holes between the
islets, probably represent the 
connecting points with the nutritional channels, which could constantly
transport nutrients to islet cells, and enable their survival in vitro survive
over a long period of time. The flask-cultured islets cells TEM and SEM showed
that there were tightly connected lesser secretory granules and vacuolar
degeneration in mitochondria in the cytoplasma. Because of stationary culture,
nourishment, gases and wastes in RCCS densities were not uniform. The cells
could be presented monolayer growth, the cell density is low, and there is no
differentiation phenomenon [3].

The constant randomization of the normal gravity vector
in the rotating process under simulated microgravity conditions subjects the
cells to a state of simulated freefall. As the cells grow in size, the rotation
speed is adjusted to compensate for the increased settling rates. Its low shear
also reduces the mechanical harm to the cells. The cells in RCCS fulfill gas
exchange by membrane diffusion in order to attain gases, nutrients and wastes
optimal transfer [4]. In addition, the cells in RCCS present some

degree of three-dimensional spatial freedom, which
promotes cell-cell and cell-matrix interactions by histology characteristic and
benefits to cell differentiation and avoids necrotic center formation [5].
These are the advantages of RCCS over the static culture.

The result of the present study implys that simulated
microgravity culture offered an ample nutrients supply for islet cells and
prolonged survival time of islet cells in vitro and that secretory
function with a better morphology when compared with flask-cultured islet
cells. This may have potential in getting enough donor islet cells in vitro for
the treatment of insulin dependent diabetes.

References

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Received:
October 13, 2003 Accepted: November 17, 2003

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author: Tel, 86-451-53643849-5870; E-mail, [email protected]