Ran XZ, Su YP, Wei YJ, Ai GP, Cheng TM, Lin Y. Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation. World J Gastroenterol 2001; 7(1): 140-142
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Dr. Xin Ze Ran, Institute of Combined Injury, PLA School of Preventive Medicine, Third Military Medical University, Chongqing 400038, China. Rxz@mail.tmmu.com.cn
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World J Gastroenterol. Feb 15, 2001; 7(1): 140-142 Published online Feb 15, 2001. doi: 10.3748/wjg.v7.i1.140
Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation
Xin Ze Ran, Yong Ping Su, Yong Jiang Wei, Guo Ping Ai, Tian Min Cheng, Yuan Lin
Xin Ze Ran, Yong Ping Su, Yong Jiang Wei, Guo Ping Ai, Tian Min Cheng, Yuan Lin, Institute of Combined Injury, PLA, School of Preventive Medicine, Third Military Medical University, Chongqing 400038, China
Xin Ze Ran, graduated from Third Military Medical University in 1980, now engaged in the research of combined radiation injuries for 20 years and having 40 papers published.
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 39100119
Correspondence to: Dr. Xin Ze Ran, Institute of Combined Injury, PLA School of Preventive Medicine, Third Military Medical University, Chongqing 400038, China. Rxz@mail.tmmu.com.cn
Telephone: 0086-23-68752278 Fax: 0086-23-68752279
Received: August 8, 2000 Revised: September 22, 2000 Accepted: September 29, 2000 Published online: February 15, 2001
Citation: Ran XZ, Su YP, Wei YJ, Ai GP, Cheng TM, Lin Y. Influencing factors of rat small intestinal epithelial cell cultivation and effects of radiation on cell proliferation. World J Gastroenterol 2001; 7(1): 140-142
Crypt epithelial cells in normal small intestine proliferate at a high speed. But they are very difficult to culture in vitro and passage stably. A lot of studies have been done[1-16]. Some domestic labs isolated and cultured crypt cells from embryonal intestines and aseptic animal intestine, but failed. We introduced normal rat epithelial cell line IEC-6 from the USA and its living condition for stable passage was successfully established after trials. The cell line was testified to be the small intestinal epithelial cell by electronmicroscopy, immunihistochemistry and enzymatic-histoch-emistry. It has been applied to some related research work[17-21]. It was found that many factors were involved in the culture system. Our present study focuses on the culture method and the influencing factors on IEC-6.
MATERIALS AND METHODS
Dulbecco’s modified Eagle Medium (DMEM), HEPES from Sigma Cooperation, 3H-TdR with the radioactive concentration of 37 MBq/mL and activity ratio of 740 GBp/mL is the product of the Chinese Nuclear Science Institute.
Carbon dioxide culture case, Model Queue 2721, USA; automatic liquid scintillation counter, Model 1217, Sweden; cell harvester, Model 2T-II, Zhejiang Province; and microplate, Japan.
Intestinal epithelial cell line, No. 6, IEC-6 was provided by the General Hospital of Massachusetts, Boston, USA.
Culture liquid medium
Liquid DMEM/L was made up according to the protocol supplemented with HEPES 10 mmol, penicillin 105 U, streptomycin 100 mg, sodium carbonate 3.7 g and fetal bovine serum 100 mmol. L-glutamine 200 mmol/L was added before use.
Establishment of cell passage and detection of activity
IEC-6 cell line was immediately put into 40 °C water bath to thaw after being taken from liquid nitrogen, centrifuged for 10 minutes at 1000 r/min. Liquid medium was added per bottle after the supernatant was deserted. Then the bottle was put into the carbon dioxide case (10% CO2, 18.6% O2 100% relative humidity, 37 °C). After the cells adhered to the wall, change the liquid once, then passage on the 5th day. When the cell was passaged or the activity was detected, the liquid medium was deserted and 0.02% EDTA 8 mL was added for digestion of 30 minutes at 37 °C. The incompletely digested cells were scraped softly with curved tube, passed into centrifuge tube and centrifuged for 8 minutes at 1000 r/min. Supernatant was deserted and the liquid medium was added to a certain concentration. The cells were then seeded onto 96-well plates and cultured for 72 hours. 3H-TdR, 1.5uCi per well, was added at the 12th hour before the culture was stopped. At the end of the culture, the cells were digested with 0.02% EDTA, harvested on the glass fiber filter membrane, and heated at 80 °C. When the membrane cooled down to the room temperature, 8 mL scintillation liquid was added, Cpm was measured with automatic liquid scintillation counter.
IEC-6 cells at various densities in microplate wells were labeled with 3H-TdR 18.5 kBq and cultured for 72 hours to investigate its effect on proliferation. Table 1 shows that at a certain range of densities, 3H-TdR incorporation increased with the IEC-6 amount, the peak was at 10 × 104/well. Positive correlation was found between cell density (X) and 3H-TdR incorporation (y) at the range of 1.25 × 104-10 × 104/well with the correlation coefficient r = 0.956 and regression equation y = 2177X + 3575 (min-1). When the cell density was more than 11.25 × 104/well, there was negative correlation (r = 0.986, y) = 36782-1253 X), possibly due to the limit amount of nutrition, liquid evaporation and subject to changes of culture condition.
Table 1 Effect of density on 3H-TdR incorporation in culture cell (x-± s).
Density (× 104/well)
Density (× 104/well)
3547 ± 681
23648 ± 1398
9941 ± 413
20593 ± 2245
17931 ± 2051
19812 ± 2310
19825 ± 2135
17638 ± 959
23789 ± 2536
14874 ± 881
Effect of culture time
Incorporation of 3H-TdR was different after IEC-6 was cultured for 6, 12, 24, 48, 72 and 96 hours (Table 2). Within 72 hours, incorporation increased from 1846 ± 146 to 25727 ± 4006 (min-1) along with the time prolongation and it reached its peak at 72 hour. But when the culture time extended to 96 hours, the incorporation decreased. This may be caused by the activity inhibition of some IEC-6 under non-physical conditions.
Table 2 Effect of culture time on IEC-6 cell proliferation (x-± s).
Culture time (h)
Culture time (h)
1846 ± 146
21258 ± 1240
4038 ± 363
25727 ± 2006
6367 ± 588
24355 ± 2079
Effect of different 3H-TdR dosage
In this study, different dosage of 3H-TdR was administered in the IEC-6 culture system. A linear correlation was found between the 3H-TdR incorporation and dosage when the dose was below 55.5 KBq/well. When larger dosage was used, the incorporations slightly increased or decreased (Table 3). The radioactive damage to cells and consequent inhibition of DNA synthesis by high concentration of 3H-TdR contributed to the incorporation decrease. Generally, the dosage of 18.5 KBq/well 3H-TdR to 10 × 104 cell yielded a satisfactory result of incorporation 2.5 × 104.
Table 3 Effect of 3H-TdR dosage on IEC-6 cell proliferation (x-± s).
3H-TdR dosage (kBq/well)
3H-TdR dosage (kBq/well)
139 ± 29
27555 ± 1637
1333 ± 118
37235 ± 1485
10136 ± 1083
41874 ± 1213
16880 ± 1447
48072 ± 1676
24890 ± 1623
42430 ± 1735
Effect of pH in lipid medium
pH of culture medium is one of the most important factors in cell culture. To optimize the culture condition, the pH value was set at 6.0, 6.6, 7.26, 7.6, 8.0 and 8.8, and 3H-TdR incorporation was measured respectively (Table 4). The incorporation was the highest at pH7.26, lower pH at 6.6 and 7.6, and the lowest at pH6.0 and 8.0. In the common sense, cells can survive when pH ranged from 6 to 8. Variant cells and animal species do not have the same optimal pH. It is believed that optimal pH has an effect on the survival of cells in vitro by adjusting the intracellular enzymes and proliferation factors. We therefore set the optimal pH7.26 in IEC-6 culture medium.
Table 4 Effect of pH of culture medium on 3H-TdR incorporation (x-± s).
4528 ± 660
12897 ± 1301
18771 ± 920
1305 ± 146
22510 ± 1448
636 ± 102
Effect of insulin and concentration of fetal bovine serum
Fetal bovine serum is one of the essential factors in cell culture in vitro. If the concentration of fetal bovine serum is too low, cells will die or have proliferation prohibited. When the concentration is too high, the osmotic pressure in culture medium will change and it will influence the survival of cells. In this study, we found that 10% of fetal bovine serum was optimal in culture medium. Content of glucose in DMEM was high (4500 mg/L) and insulin can speed up glucose oxygenolysis and transportation through cell membrane, so the use of glucose was accelerated in the cells. The results showed that incorporation of 3H-TdR was higher in cells treated with insulin than in the cells (Table 5) without insulin treatment.
Table 5 Effect of insulin and concentration of fetal bovine serum on IEC-6 cell proliferation (x-± s).
Fetal bovine serum (%)
476 ± 22
510 ± 101
13111 ± 978
1901 ± 580
14756 ± 1094
9097 ± 1069
20262 ± 2012
14569 ± 1136
23666 ± 1114
18775 ± 1361
22743 ± 1728
17645 ± 1289
22590 ± 1603
16965 ± 1147
To investigate the experimental method, stability and the researcher’s error, repeatability was measured by dividing the same culture system of IEC-6 into 30 parts. The incorporation of 3H-TdR was 24327 ± 808 (min-1). The value ranged from 23921 to 24733 when P < 0.01 and coefficient of variation was 3.32%.
Effect of ionizing radiation on IEC-6
Intestinal epithelial cells are sensitive to ionizing radiation. The changes of incorporation of 3H-TdR showed the damage of ionizing radiation on cells which reflected the cell biological characteristics. When IEC-6 was not exposed to radiation, the incorporation was 24327 ± 808. Incorporation after 4Gy, 8Gy, 16Gy, 2Gy and 26y irradiation were 31.8%, 24.1%, 15.2%, 11.2% and 8.3% of control. Significantly negative dose-effect relation was found with the relative coefficient r = -0.970 (Table 6).
Table 6 Effect of ionizing radiation dosage on 3H-TdR incorporation in IEC-6 cell (x-± s).
13427 ± 803
3698 ± 371
7736 ± 765
3381 ± 235
7249 ± 472
3041 ± 327
5863 ± 594
2725 ± 348
4865 ± 586
2481 ± 263
4136 ± 424
2019 ± 154
r = -0.970
In summary, methods of IEC-6 culture, passage and activity detection established in this study have the advantage of easy handling, being reliable in results, using less amounts of cells and a good repeatability. Subjective error can be avoided in measurement of epithelial proliferation with radioactivity. These will provide an ideal method for the research[23-35] on intestinal epithelimm[36-42].
Edited by Ma JY
Rogler G, Aschenbrenner E, Gross V, Stange EF, Schölmerich J. Intracellular transport of high-density lipoprotein 3 in intestinal epithelial cells (Caco-2) is tubulin associated.Digestion. 2000;61:47-58.
Hofman P, Piche M, Far DF, Le Negrate G, Selva E, Landraud L, Alliana-Schmid A, Boquet P, Rossi B. Increased Escherichia coli phagocytosis in neutrophils that have transmigrated across a cultured intestinal epithelium.Infect Immun. 2000;68:449-455.
Said HM. Cellular uptake of biotin: mechanisms and regulation.J Nutr. 1999;129:490S-493S.
Eckmann L, Stenson WF, Savidge TC, Lowe DC, Barrett KE, Fierer J, Smith JR, Kagnoff MF. Role of intestinal epithelial cells in the host secretory response to infection by invasive bacteria. Bacterial entry induces epithelial prostaglandin h synthase-2 expression and prostaglandin E2 and F2alpha production.J Clin Invest. 1997;100:296-309.
Tripuraneni J, Koutsouris A, Pestic L, De Lanerolle P, Hecht G. The toxin of diarrheic shellfish poisoning, okadaic acid, increases intestinal epithelial paracellular permeability.Gastroenterology. 1997;112:100-108.
Augustijns PF, Borchardt RT. Transport and metabolism of delta sleep-inducing peptide in cultured human intestinal epithelial cell monolayers.Drug Metab Dispos. 1995;23:1372-1378.
Zheng L, Chen J, Zhu Y, Yang H, Elmquist W, Hu M. Comparison of the transport characteristics of D- and L-methionine in a human intestinal epithelial model (Caco-2) and in a perfused rat intestinal model.Pharm Res. 1994;11:1771-1776.
Chikhale PJ, Borchardt RT. Metabolism of L-alpha-methyldopa in cultured human intestinal epithelial (Caco-2) cell monolayers. Comparison with metabolism in vivo.Drug Metab Dispos. 1994;22:592-600.
Das S, Traynor-Kaplan A, Kachintorn U, Aley SB, Gillin FD. GP49, an invariant GPI-anchored antigen of Giardia lamblia.Braz J Med Biol Res. 1994;27:463-469.
Rokutan K, Sakai A, Teramoto F, Kido Y, Shizuka F, Kishi K. Epidermal growth factor-induced mitogen signals in cultured intestinal epithelial cells.J Gastroenterol. 1994;29 Suppl 7:59-62.
Lambert RW, Kelleher RS, Wickham LA, Vaerman JP, Sullivan DA. Neuroendocrinimmune modulation of secretory component production by rat lacrimal, salivary, and intestinal epithelial cells.Invest Ophthalmol Vis Sci. 1994;35:1192-1201.
Sunitha I, Meighen DL, Hartman DP, Thompson EW, Byers SW, Avigan MI. Hepatocyte growth factor stimulates invasion across reconstituted basement membranes by a new human small intestinal cell line.Clin Exp Metastasis. 1994;12:143-154.
Sanderson IR, He Y. Nucleotide uptake and metabolism by intestinal epithelial cells.J Nutr. 1994;124:131S-137S.
Quaroni A, Wands J, Trelstad RL, Isselbacher KJ. Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.J Cell Biol. 1979;80:248-265.
Gike M, Kanai M, Lynch-Devaney K, Podolsky DK. Rapid mitogen-activated protein kinase activation by transforming growth factor in wounded rat intestinal epithelial cells.World J Gastroenterol. 1998;4:263.
Wang XH, Zhou Z, Zhu GX, Lou SF, Ran XZ, Cheng TM, Yu ZP. Protective effect of keratinocyte growth factor on intestinal epithelial cell line No.6 after irradiation.Disan Junyi Daxue Xuebao. 2000;22:713-715.
Bai XD, Liu XH, Su YP. Inhibitory effects intestinal mucus on bacterial adherence to cultured intestinal epithelial cell after burns.Disan Junyi Daxue Xuebao. 1998;20:214-216.
Zhang WJ, Ke JX, Shi TZ, L¨1 YH, Su YP, Bai XD, Ran XZ. An improving method of ultramicrotomy for cultured cell.Disan Junyi Daxue Xuebao. 1996;18:A48-A49.
Wang JP, Hu CM, Su YP, Cheng TM. Preliminary identification of the genes expressed in intestinal epithelial cell after radiation injury in mice.Disan Junyi Daxue Xuebao. 1999;21:387-389.
Xu H, Cheng TM, Su YP, Lin Y. Effects of total body irradiation on functions of small intestinal intraepithelial lymphocytes.Zhonghua Fangshe Yixue Yu Fanghu Zazhi. 1999;19:18-21.
Zhou DH, Shen YS, Zhao MR. The application of MTT colorimetric aeasured the proliferation of lymphocytes and activity of rat/mouse IL-2.Zhongguo Mianyixue Zazhi. 1986;2:39-44.
Zhang XL, Wang GH, Han L, Su L, Sheng Z, Zou JH, Liu JY. Transforma-tion and influencing factors of the peripheral blood T lymphocytes of dometic rabbits.Beijing Yike Daxue Xuebao. 1988;20:10.
Peng ZS, Liang ZC, Liu MC, Ouyang NT. Studies on gastric epithelial cell proliferation and apoptosis in Hp associated gastric ulcer.Shijie Huaren Xiaohua Zazhi. 1999;7:218-219.
Wu YZ, Wu JS, Lai DN, Ma QJ, He ZS, Gao DM. Relationship between ectasias in gastric microvascular system and cytokinetics in gastric mucous epithelial cell group in PHG-MH rats.Huaren Xiaohua Zazhi. 1998;6:752-754.
Fang DC, Liu W, Lang HJ, Liu WW. Effects of Naa-2SeOa-3 on UnscheduledDNA synthesis, lipid peroxidation and ras P21 expression in gastric epithelial cells.Huaren Xiaohua Zazhi. 1998;6:421-422.
Wang LD, Zhou Q, Gao SS, Li YX, Yang WC. Measurements of cell prolifera-tion in esophageal and gastric cardia epithelia of subjects in a high incidence area for esophageal cancer.China Natl J New Gastroenterol. 1996;2:82-85.
Chen XM, Han DW, Noguchi K, Tanikawa K. Uptake of bacterial lipopolysac-charide and expression of tumor necrosis factor-mRNA in isolated rat intrahe-patic bile duct epithelial cells.China Natl J New Gastroenterol. 1997;3:3-5.
Wang ZX, Shen HF, Chen HJ. Adherent properties of Helicobacter pylori to human epithelial cells.China Natl J New Gastroenterol. 1997;3:35-37.
Indaram AV, Nandi S, Weissman S, Lam S, Bailey B, Blumstein M, Greenberg R, Bank S. Elevated basal intestinal mucosal cytokine levels in asymptomatic first-degree relatives of patients with Crohn's disease.World J Gastroenterol. 2000;6:49-52.
Lai YC, Yang SS, Wu CH, Chen TK. Endoscopic hemoclip treatment for bleeding peptic ulcer.World J Gastroenterol. 2000;6:53-56.
Pan QS, Fang ZP, Zhao YX. Immunocytochemical identification and localization of APUD cells in the gut of seven stomachless teleost fishes.World J Gastroenterol. 2000;6:96-101.
Li YX, Li JS, Li N. Improved technique of vascular anastomosis for small intestinal transplantation in rats.World J Gastroenterol. 2000;6:259-262.
Liu BH, Chen HS, Zhou JH, Xiao N. Effects of endotoxin on endothelin receptor in hepatic and intestinal tissues after endotoxemia in rats.World J Gastroenterol. 2000;6:298-300.
Fu XB, Yang YH, Sun TZ, Gu XM, Jiang LX, Sun XQ, Sheng ZY. Effect of intestinal ischemia-reperfusion on expressions of endogenous basic fibroblast growth factor and transforming growth factor betain lung and its relation with lung repair.World J Gastroenterol. 2000;6:353-355.
Wang QG, He LY, Chen YW, Hu SL. Enzymohistochemical study on burn effect on rat intestinal NOS.World J Gastroenterol. 2000;6:421-423.
Chen XM, LaRusso NF. Human intestinal and biliary cryptosporidiosis.World J Gastroenterol. 1999;5:424-429.
Zhou Q, Xu TR, Fan QH, Zhen ZX. Clinicopathologic study of primary intestinal B cell malignant lymphoma.World J Gastroenterol. 1999;5:538-540.
Huang B, Wu ZB, Ruan YB. Expression of nm23 gene in hepatocellular carcinoma tissue and its relation with metastasis.World J Gastroenterol. 1998;4:266-267.
Xu CT, Pan BR, Wang YM, Zhang RY, Substance P. Vasoactive intestinal peptide and leu-enkephalin in plasma and gastric juice of patients with precancer-ous lesions and gastric cancer.China Natl J New Gastroenterol. 1995;1:27-29.
Xu CT, Wang RL, Pan BR. Endoscopic evaluation of gastrointestinal tract lesions in patients with iron-deficiency anemia.China Natl J New Gastroenterol. 1996;2:95-98.