Safety Evaluation of Human Fibroblasts in Mice: Tumorigenicity, 13-week Toxicity and Distribution Studies

Hyung Jun Choi; Euna Kwon; Jeong-Hee Sohn; Jeong-Hwan Che; Kook Hyun Lee; Jong Wan Kim; Jaydo Choi; Byeong-Cheol Kang

doi:10.5625/lar.2010.26.2.181

Journal List > Lab Anim Res > v.26(2) > 1053606

Go to TopGo to Top Go to BottomGo to Bottom

TOOLS

Choi, Kwon, Sohn, Che, Lee, Kim, Choi, and Kang: Safety Evaluation of Human Fibroblasts in Mice: Tumorigenicity, 13-week Toxicity and Distribution Studies

Original Article

Lab. Anim. Res. 2010;26(2):181-196.

Published online: 17 June 2010

DOI: https://doi.org/10.5625/lar.2010.26.2.181

Safety Evaluation of Human Fibroblasts in Mice: Tumorigenicity, 13-week Toxicity and Distribution Studies

Hyung Jun Choi^1,², Euna Kwon¹, Jeong-Hee Sohn¹, Jeong-Hwan Che¹, Kook Hyun Lee¹, Jong Wan Kim³, Jaydo Choi³, Byeong-Cheol Kang^1,^2,^∗

¹Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea

²Department of Immunology and Laboratory Animal Medicine, College of Medicine, Seoul National University, Seoul, Korea

³S.BIOMEDICS Co., Ltd. Seoul, Korea

^∗Corresponding author: Byeong-Cheol Kang, Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, 28 Yongon-dong, Jongno-gu, Seoul 110-744, Korea Tel: +82-2-2072-0841 Fax: +82-2-741-7620 E-mail: bckang@snu.ac.kr

Received 26 November 200919 March 2010 Accepted 14 June 2010

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Human fibroblasts were developed for cellular therapy with the aim of correcting of depressed scars, but the safety of that in vivo is unclear. In this study, we assessed the safety of human fibroblasts by investigating the tumorigenicity, 13-week toxicity and through distribution studies. In the tumorigenicity test, nude mice were divided into three dosage level treatment groups with a negative/positive control group. At 6 months after intradermal transplantation, all of the treatment groups showed no development of a nodule on the injection sites and organs. Toxicity studies were performed using ICR and BALB/c mice for 13 weeks. The mice were divided into three dosage level treatment groups with a control and a syngeneic group. There was no treatment-related effect on clinical signs, mortality, body weight, food/ water consumption, hematology, serum biochemistry, urine, necropsy findings and histopathological findings in any groups. These results suggest that the no-observed-effect level (NOEL) of the human fibroblasts was greater than 7.5×10⁷ cells/kg for mice. In the distribution study, groups were treated with fibroblasts labeled with a fluorescent dye (CM-DiI) at low and high doses with a control and a syngeneic group. At 24 hours, a large percentage of the labeled fibroblasts were observed at the dermal layer. At 3 months, fluorescence of the labeled fibroblasts continued to be observed. Other tissues were not detected the fluorescence at any time. These studies demonstrate that the safety of human fibroblasts is reasonable with no toxic effect, no tumorigenicity and retention in the dermis. Our studies define preclinical safety testing standards relevant to the development of cellular therapeutics.

Keywords: Fibroblast, tumorigenicity, toxicity, distribution, cell therapy

REFERENCES

Adonai N.., Nguyen K.N.., Walsh J.., Iyer M.., Toyokuni T.., Phelps M.E.., McCarthy T.., McCarthy D.W.., Gambhir S.S.2002. Ex vivo cell labeling with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for imaging cell trafficking in mice with positron-emission tomography. Proc. Natl. Acad. Sci. U.S.A. 99(5):3030–3035.

Allers C.., Sierralta W.D.., Neubauer S.., Rivera F.., Minguell J.J.., Conget P.A.2004. Dynamic of distribution of human bone marrow-derived mesenchymal stem cells after transplantation into adult unconditioned mice. Transplantation. 78(4):503–508.

Benten D.., Cheng K.., Gupta S.2006. Identification of transplanted human cells in animal tissues. Methods Mol. 326:189–201.

Boss W.K. Jr.., Usal H.., Chernoff G.., Keller G.S.., Lask G.P.., Fodor P.B.2000. Autologous cultured fibroblasts as cellular therapy in plastic surgery. Clin. Plast. Surg. 27(4):613–626.

Bulte J.W.., Duncan I.D.., Frank J.A.2002. In vivo magnetic resonance tracking of magnetically labeled cells after transplantation. J. Cereb. Blood Flow Metab. 22(8):899–907.

Cho J.J.., Malhi H.., Wang R.., Joseph B.., Ludlow J.W.., Susick R.., Gupta S.2002. Enzymatically labeled chromosomal probes for in situ identification of human cells in xenogeneic transplant models. Nat. Med. 8(9):1033–1036.

Ferrari A.., Hannouche D.., Oudina K.., Bourguignon M.., Meunier A.., Sedel L.., Petite H.2001. In vivo tracking of bone marrow fibroblasts with fluorescent carbocyanine dye. J. Biomed. Mater. Res. 56(3):361–367.

Fischer U.M.., Harting M.T.., Jimenez F.., Monzon-Posadas W.O.., Xue H.., Savitz S.I.., Laine G.A.., Cox, C.S. Jr.2009. Pulmonary passage is a major obstacle for intravenous stem cell delivery: the pulmonary first-pass effect. Stem Cells Dev. 18(5):683–692.

Galbraith D.N.2004. Regulatory and microbiological safety issues surrounding cell and tissue-engineering products. Biotechnol. Appl. Biochem. 40(Pt 1), 35-39. Guerret, S., Govignon, E., Hartmann, D.J. and Ronfard, V. (2003) Longterm remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice: immuno-localization of human cells and characterization of extracellular matrix. Wound Repair Regen. 11(1):35–45.

Hebda P.A.., Dohar J.E.1999. Transplanted fetal fibroblasts: survival and distribution over time in normal adult dermis compared with autogenic, allogenic, and xenogenic adult fibroblasts. Otolaryngol Head Neck Surg. 121(3):245–251.

Kovacsovics-Bankowski M.., Mauch K.., Raber A.., Streeter P.R.., Deans R.J.., Maziarz R.T.., Van't Hof W.2008. Preclinical safety testing supporting clinical use of allogeneic multipotent adult progenitor cells. Cytotherapy. 10(7):730–742.

Lawrenz B.., Schiller H.., Willbold E.., Ruediger M.., Muhs A.., Esser S.2004. Highly sensitive biosafety model for stem-cell-derived grafts. Cytotherapy. 6(3):212–222.

Lee E.S.., Kwon E.A.., Park J.R.., Kang B.C.., Kang K.S.., Cho M.H.2007. Tumorigenesis study of canine adipose derived-mesenchymal stem cell. J. Toxicol. Pub. Health. 23(3):271–278.

Lee J.H.., Kim Y.K.., Lee S.J.2006. Treatment of Aplasia Cuti Congenita using allogenic dermal matrix and cultured epithelial autograft: A Case Report. J. Korean Soc. Plast. Reconstr. Surg. 33(5):672–675.

Liu L.., Sun Z.., Chen B.., Han Q.., Liao L.., Jia M.., Cao Y.., Ma J.., Sun Q.., Guo M.., Liu Z.., Ai H.., Zhao R.C.2006. Ex vivo expansion and in vivo infusion of bone marrow-derived Flk-1+CD31-CD34- mesenchymal stem cells: feasibility and safety from monkey to human. Stem Cells Dev. 15(3):349–357.

Meyerrose T.E.., De Ugarte D.A.., Hofling A.A.., Herrbrich P.E.., Cordonnier T.D.., Shultz L.D.., Eagon J.C.., Wirthlin L.., Sands M.S.., Hedrick M.A.., Nolta J.A.2007. In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models. Stem Cells. 25(1):220–227.

Moeller F.., Nielsen F.C.., Nielsen L.B.2003. New tools for quantifying and visualizing adoptively transferred cells in recipient mice. J. Immunol. Methods 282(1-2), 73-82. Rubio, D., Garcia-Castro, J., Martin, M.C., de la Fuente, R., Cigudosa, J.C., Lloyd, A.C. and Bernad, A. (2005) Spontaneous human adult stem cell transformation. Cancer Res. 65(8):3035–3039.

Sandulache V.C.., Zhou Z.., Sherman A.., Dohar J.E.., Hebda P.A.2003. Impact of transplanted fibroblasts on rabbit skin wounds. Arch. Otolaryngol. Head Neck Surg. 129(3):345–350.

Schrepfer S.., Deuse T.., Reichenspurner H.., Fischbein M.P.., Robbins R.C.., Pelletier M.P.2007. Stem cell transplantation: the lung barrier. Transplant. Proc. 39(2):573–576.

Seo S.W.., Chang C.H.., Cho M.S.., Hong Y.G.., Jeon S.W.2007. Treatment of partial thickness skin defect with cultured allogenic keratinocytes (Kaloderm.). J. Korean Soc. Traumatol. 20(1):1–5.

Thompson M.., Wall D.M.., Hicks R.J.., Prince H.M.2005. In vivo tracking for cell therapies. Q. J. Nucl. Med. Mol. Imaging. 49(4):339–348.

Trosko J.E.., Tai M.H.2006. Adult stem cell theory of the multi-stage, multi-mechanism theory of carcinogenesis: role of inflammation on the promotion of initiated stem cells. Contrib. Microbiol. 13:45–65.

Vilalta M.., Degano I.R.., Bago J.., Gould D.., Santos M.., Garcia-Arranz M.., Ayats R.., Fuster C.., Chernajovsky Y.., Garcia-Olmo D.., Rubio N.., Blanco J.2008. Biodistribution, longterm survival, and safety of human adipose tissue-derived mesenchymal stem cells transplanted in nude mice by high sensitivity non-invasive bioluminescence imaging. Stem Cells Dev. 17(5):993–1003.

Watson D.., Keller G.S.., Lacombe V.., Fodor P.B.., Rawnsley J.., Lask G.P.1999. Autologous fibroblasts for treatment of facial rhytids and dermal depressions. A pilot study. Arch. Facial Plast. Surg. 1(3):165–170.

Yoon S.H.., Shim J.S.., Jung J.M.., Park D.H.., Song C.H.2008. The usefulnesss of cultured allogenic keratinocyte for burn treatment. J. Korean Soc. Plast. Reconstr. Surg. 35(4):413–418.

Figure 1.

Tumor incidence rate of female BALB/c-nu mice intradermally treated with human fibroblasts in the tumorigenicity study.

Figure 2.

Body weight changes of male (left chart) and female (right chart) mice intradermally treated with human fibroblasts in the 13-week toxicity study.

Figure 3.

Microscopic findings of ICR mice intradermally treated with human fibroblasts in the 13-week toxicity study. (A) Deciduoma in uterus. ×40, (B) Granulomatous inflammation in the dermis. Note the needle-like crystals and necrosis. ×40, (C) Centrilobular vacuolation in liver. ×40. H&E.

Figure 4.

Photomicrographs of the skin and other organs of mice Intradermally treated with labeled human fibroblasts in the distribution study. The images from the first layer to the third layer are each skin sample at 24 hours, 4 weeks and 13 weeks (×100). The images of the fourth layer are other organs that were not detected with the labeled fibroblasts (×40).

Table 1.

Summary of the hematological values of mice intradermally treated with human fibroblasts in the 13-week toxicity study

	Dosage in (cells/kg B.W.) Items	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (Syngeneic)
Male	Leukocytes (10³/µL)	3.32±1.479	4.96±0.479	2.85±0.969	3.39±1.316	2.16±0.412
	Erythrocytes (10⁶/µL)	7.8±0.43	7.9±0.19	7.6±0.47	8.2±0.59	8.9±0.62
	Hemoglobin (g/dL)	11.2±0.74	11.5±0.20	11.0±0.41	11.9±1.03	12.8±1.03
	Hematocrit (%)	33.7±2.18	35.6±0.69	33.9±1.90	37.0±4.07	42.1±3.11
	Platelets (10³/µL)	953±140.0	1087±78.6	945±65.3	1018±83.3	669±131.5
	MCV (fl)	43.6±3.11	45.4±1.43	44.5±1.57	45.3±2.75	47.6±0.63
	MCH (pg)	14.4±0.18	14.7±0.45	14.5±0.43	14.6±0.54	14.4±0.54
	MCHC (g/dL)	33.2±2.40	32.4±0.73	32.6±0.64	32.3±1.30	30.3±0.80
	Neutrophils (%)	6.7±1.56	7.4±2.06	12.7±5.84	10.0±4.10	12.4±5.73
	Eosinophils (%)	3.1±1.99	1.2±0.56	2.8±0.77	2.6±1.11	5.4±2.80
	Basophils (%)	0.4±0.11	0.3±0.12	0.3±0.12	0.3±0.12	0.4±0.09
	Lymphocytes (%)	83.5±2.51	85.5±1.62	76.4±6.49	79.9±5.49	74.2±4.19
	Monocytes (%)	4.7±0.92	4.4±0.32	5.7±0.76	5.5±1.75	5.6±1.25
Fem ale	Leukocytes (10³/µL)	2.67±1.006	5.42^a±1.037	3.77±1.805	2.55±0.733	3.95±1.482
	Erythrocytes (10⁶/µL)	7.7±0.24	7.7±0.37	7.9±0.29	8.0±0.64	8.4±0.32
	Hemoglobin (g/dL)	11.2±0.05	11.7±0.68	11.7±0.81	11.5±1.23	12.4±0.53
	Hematocrit (%)	32.9±1.83	35.7±3.05	35.9±2.30	36.7±3.43	39.1±1.09
	Platelets (10³/µL)	896±144.6	869±131.3	1011±98.7	989±115.1	638±28.6
	MCV (fl)	42.7±3.25	46.4±1.94	45.1±2.32	46.0±1.16	46.6±0.62
	MCH (pg)	14.5±0.45	15.3±0.30	14.7±0.75	14.4±0.46	14.7±0.11
	MCHC (g/dL)	34.0±1.84	32.9±1.08	32.7±0.49	31.3^a±0.80	31.7±0.64
	Neutrophils (%)	8.4±5.42	8.2±2.16	9.6±2.10	10.3±2.83	13.1±1.81
	Eosinophils (%)	3.0±1.71	1.1^a±0.24	1.5±0.67	2.0±0.67	2.3±1.21
	Basophils (%)	0.3±0.13	0.4±0.16	0.4±0.11	0.2±0.09	0.3±0.11
	Lymphocytes (%)	81.0±4.63	84.6±3.09	82.1±3.38	79.8±3.55	77.1±2.15
	Monocytes (%)	5.5±0.83	4.6±1.17	5.2±1.00	6.3±1.23	5.5±0.91

All values are expressed as mean±SD, n=5

^a Significantly different from the control group; P<0.05.

Table 2.

Summary of the serum biochemical values of mice intradermally treated with human fibroblasts in the 13-week toxicity study

	Dosage in (cells/kg B.W.) Items	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (Syngeneic)
Male	Urea Nitrogen (mg/dL)	27.9±6.02	29.8±5.06	29.7±3.74	28.5±3.05	28.9±4.36
	Cholesterol (mg/dL)	145±26.8	131±27.8	149±23.9	144±23.4	130±14.8
	Total Protein (g/dL)	4.7±0.28	4.7±0.35	4.8±0.25	4.7±0.11	4.8±0.10
	Albumin (g/dL)	1.5±0.07	1.5±0.14	1.6±0.09	1.5±0.08	1.6±0.03
	Total Bilirubin (mg/dL)	0.1±0.06	0.1±0.04	0.1±0.05	0.1±0.04	0.0±0.04
	Alkaline Phosphatase (IU/L)	37±10.5	38±11.1	35±7.60	40±17.1	83±6.00
	Aspartate Aminotransferase (IU/L)	54±16.9	48±13.5	49±9.0 0	41±4.9 0	54±29.9
	Alanine Aminotransferase (IU/L)	25±10.0	19±4.90	20±5.80	17^a±2.20	29±9.70
	Creatinine (mg/dL)	0.5±0.17	0.4±0.08	0.4±0.06	0.4±0.05	0.3±0.05
	Triglycerides (mg/dL)	67±41.0	45±18.4	52±18.0	50±23.3	62±27.7
	Glucose (mg/L)	173±35.9	170±29.4	178±19.2	187±30.4	173±15.8
	A/G ratio	0.5±0.03	0.5±0.06	0.5±0.03	0.5±0.03	0.5±0.00
	Potassium (mmol/L)	5.65±0.367	5.76±0.527	5.62±0.379	5.85±0.568	5.75±0.355
	Chlorine (mmol/L)	109.3±3.25	111.9±2.68	113.0^a±1.91	113.2^a±1.48	119.5±1.15
	Calcium (mg/dL)	8.3±0.29	8.5±0.22	8.4±0.24	8.2±0.29	8.4±0.23
	Phosphorus (mg/dL)	6.5±0.66	6.0±0.86	6.4±0.70	6.4±0.53	5.7±0.76
	Sodium (mmol/L)	149.9±2.39	150.4±1.03	151.5±0.95	152.0^a±1.89	153.9±1.12
Fem ale	Urea Nitrogen (mg/dL)	23.3±4.23	25.3±5.29	23.0±5.67	23.2±5.49	23.9±4.47
	Cholesterol (mg/dL)	90±16.3	93±19.7	101±13.7	102±15.5	89±8.00
	Total Protein (g/dL)	4.8±0.17	4.8±0.14	4.9±0.18	5.0^a±0.20	4.7±0.15
	Albumin (g/dL)	1.6±0.06	1.6±0.07	1.7±0.08	1.6±0.07	1.6±0.08
	Total Bilirubin (mg/dL)	0.0±0.05	0.0±0.05	0.0±0.05	0.1±0.05	0.0±0.03
	Alkaline Phosphatase (IU/L)	66±11.3	80±31.2	61±13.8	70±20.2	92±4.7
	Aspartate Aminotransferase (IU/L)	60±8.80	57±8.10	54±5.20	57±10.9	80±28.2
	Alanine Aminotransferase (IU/L)	18±2.20	20±3.10	17±2.90	17±2.00	38±19.1
	Creatinine (mg/dL)	0.47±0.07	0.39^a±0.06	0.40^a±0.05	0.44±0.05	0.47±0.13
	Triglycerides (mg/dL)	67±21.5	75±48.6	79±48.4	63±30.4	86±29.7
	Glucose (mg/L)	182±15.1	198±36.6	203±24.8	189±24.7	181±35.7
	A/G ratio	0.5±0.00	0.5±0.00	0.5±0.00	0.5±0.00	0.5±0.00
	Potassium (mmol/L)	4.83±0.302	5.11±0.212	5.29^a±0.465	5.33^a±0.349	5.55±0.369
	Chlorine (mmol/L)	115.0±2.10	116.8±2.78	115.9±1.88	115.0±1.68	120.7±1.48
	Calcium (mg/dL)	8.1±0.35	8.3±0.12	8.1±0.23	8.0±0.42	8.1±0.21
	Phosphorus (mg/dL)	6.4±1.76	6.0±0.99	6.3±0.96	6.9±0.69	6.2±0.83
	Sodium (mmol/L)	150.0±1.32	149.7±1.76	150.0±1.62	150.9±1.79	152.1±1.90

All values are expressed as mean±SD, n=10

^a Significantly different from the control group; P<0.05.

Table 3.

Summary incidence of the gross findings of mice intradermally treated with human fibroblasts in the 13-week toxicity study

Sex Dosage in (cells/kg B.W.) Tissue/Observation	Male					Female
Sex Dosage in (cells/kg B.W.) Tissue/Observation	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (Syngeneic)	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (Syngeneic)
Skin
scab around genitalia	0	2	0	0	0	0	0	0	0	0
Seminal vesicle/Coagulation gland
atrophy	0	1	0	0	0	0	0	0	0	0
Kidney
cyst	1	0	0	0	0	0	0	0	0	0
atrophy	1	0	0	0	0	0	0	0	0	0
Preputial gland
cyst	1	1	0	0	0	0	0	0	0	0
Mammary gland
subcutaneous, mass	0	1	0	0	0	0	0	0	0	0
Uterus
uterine horn, cyst	0	0	0	0	0	0	1	0	0	0
Ovary
cyst	0	0	0	0	0	0	1	1	0	0

Table 4.

Summary of the absolute organ weight values of mice intradermally treated with human fibroblasts in the 13-week toxicity study

	Dosage in (cells/kg B.W.) Items	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10₇	7.5x10⁷ (Syngeneic)
Male	Necropsy B.W.	40.762±4.00290	41.344±3.65930	44.974±3.65430	42.974±2.03980	30.104±1.14360
	Liver	1.7901±0.16666	1.7511±0.27671	1.8204±0.22916	1.7679±0.18372	1.2883±0.07363
	Spleen	0.1055±0.01088	0.1369±0.10339	0.1171±0.01905	0.1218±0.03607	0.1047±0.00763
	Kidney (R)	0.3868±0.03910	0.3332±0.04844	0.3847±0.04270	0.3762±0.07540	0.2558±0.01737
	Kidney (L)	0.3397±0.10191	0.3352±0.04346	0.3772±0.04318	0.3754±0.05737	0.2552±0.02100
	Adrenal gland (R)	0.0022±0.00078	0.0018±0.00029	0.0014^a±0.00036^a	0.0023±0.00087	0.0016±0.00037
	Adrenal gland (L)	0.0019±0.00067	0.0017±0.00051	0.0018±0.00054	0.0019±0.00033	0.0017±0.00054
	Testis (R)	0.1227±0.01143	0.1356±0.03263	0.1279±0.02309	0.1410±0.02902	0.0869±0.00636
	Testis (L)	0.1166±0.01088	0.1336±0.02572	0.1220±0.02002	0.1340±0.02722	0.0871±0.00601
	Thymus	0.0265±0.00761	0.0236±0.00891	0.0252±0.00560	0.0264±0.00644	0.0329±0.00628
	Heart	0.2007±0.01231	0.1952±0.01432	0.2072±0.01557	0.2116±0.01665	0.1601±0.00675
	Lung	0.2187±0.01975	0.2200±0.02618	0.2294±0.02111	0.2240±0.03275	0.1739±0.00676
	Brain	0.5278±0.03416	0.5284±0.02479	0.5207±0.02861	0.5158±0.03345	0.4621±0.01730
	Pituitary gland	0.0022±0.00119	0.0019±0.00078	0.0023±0.00077	0.0025±0.00065	0.0011±0.00043
Fem ale	Necropsy B.W.	29.766±2.14880	31.732±4.71320	32.533±4.24960	29.952±1.65750	22.826±0.81650
	Liver	1.5100±0.18402	1.4322±0.25739	1.4174±0.24199	1.2905±0.15086	1.0378±0.10895
	Spleen	0.1267±0.02137	0.1127±0.02482	0.1143±0.01428	0.1107±0.01865	0.1043±0.00845
	Kidney (R)	0.2344±0.02964	0.2211±0.02669	0.2072±0.02336	0.2013^a±0.02411^a	0.1532±0.01738
	Kidney (L)	0.2319±0.02718	0.2200±0.02989	0.2056±0.02367	0.1937^a±0.02295^a	0.1521±0.01735
	Adrenal gland (R)	0.0032±0.00078	0.0034±0.00058	0.0034±0.00081	0.0032±0.00081	0.0038±0.00046
	Adrenal gland (L)	0.0034±0.00102	0.0036±0.00095	0.0036±0.00082	0.0036±0.00102	0.0038±0.00051
	Ovary (R)	0.0141±0.00378	0.0137±0.00429	0.0164±0.00954	0.0129±0.00291	0.0087±0.00226
	Ovary (L)	0.0134±0.00471	0.0137±0.00452	0.0140±0.00296	0.0128±0.00264	0.0090±0.00186
	Thymus	0.0314±0.00655	0.0307±0.00858	0.0363±0.00957	0.0328±0.00830	0.0302±0.00515
	Heart	0.1548±0.01067	0.1483±0.01253	0.1534±0.01285	0.1436±0.00928	0.1217±0.00795
	Lung	0.2033±0.03259	0.2024±0.01867	0.2107±0.02600	0.1897±0.01596	0.1683±0.03073
	Brain	0.5660±0.02970	0.5328±0.03978	0.5164^a±0.01997^a	0.5243^a±0.03318^a	0.4573±0.02700
	Pituitary gland	0.0021±0.00079	0.0025±0.00101	0.0027±0.00082	0.0026±0.00051	0.0016±0.00079

All values are expressed as mean±SD, n=10

^a Significantly different from the control group; P<0.05.

Table 5.

Summary of the relative organ weight values of mice intradermally treated with human fibroblasts in the 13-week toxicity study

	Dosage in (cells/kg B.W.) Items	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (Syngeneic)
Male	Liver	4.4074±0.37572	4.2472±0.68045	4.0524±0.42968	4.1148±0.39717	4.2811±0.21926
	Spleen	0.2615±0.03979	0.3333±0.25253	0.2616±0.04547	0.2846±0.08752	0.3482±0.02867
	Kidney (R)	0.9580±0.14322	0.8092±0.12505	0.8599±0.11004	0.8762±0.17409	0.8512±0.07282
	Kidney (L)	0.8234±0.23827	0.8139±0.10962	0.8418±0.10106	0.8754±0.14099	0.8487±0.07948
	Adrenal gl. (R)	0.0053±0.00210	0.0045±0.00083	0.0031^a±0.00088^a	0.0054±0.00199	0.0052±0.00130
	Adrenal gl. (L)	0.0047±0.00174	0.0043±0.00136	0.0040±0.00108	0.0045±0.00074	0.0055±0.00179
	Testis (R)	0.3029±0.03427	0.3297±0.08319	0.2849±0.05186	0.3282±0.06661	0.2886±0.01859
	Testis (L)	0.2875±0.03110	0.3249±0.06666	0.2723±0.04779	0.3116±0.06120	0.2894±0.02088
	Thymus	0.0643±0.01552	0.0570±0.01997	0.0559±0.01079	0.0609±0.01226	0.1096±0.02202
	Heart	0.4973±0.06634	0.4745±0.04620	0.4628±0.04435	0.4929±0.03958	0.5321±0.02012
	Lung	0.5388±0.04699	0.5331±0.05693	0.5120±0.05202	0.5216±0.07454	0.5779±0.01863
	Brain	1.3031±0.12197	1.2847±0.10116	1.1638^a±0.10608^a	1.2024±0.08977	1.5366±0.07300
	Pitui gl.	0.0056±0.00296	0.0046±0.00166	0.0052±0.00173	0.0058±0.00147	0.0036±0.00147
Fem ale	Liver	5.0767±0.53478	4.5122^a±0.46906^a	4.3447^a±0.33141^a	4.3048^a±0.40825^a	4.5397±0.36554
	Spleen	0.4246±0.05509	0.3569^a±0.07185^a	0.3538^a±0.04581^a	0.3690±0.05488	0.4566±0.02726
	Kidney (R)	0.7899±0.10581	0.7030±0.08251	0.6453^a±0.10106^a	0.6718^a±0.06802^a	0.6705±0.06607
	Kidney (L)	0.7822±0.10155	0.6986±0.08591	0.6385^a±0.08979^a	0.6467^a±0.06338^a	0.6655±0.06240
	Adrenal gl. (R)	0.0108±0.00250	0.0109±0.00282	0.0107±0.00274	0.0109±0.00320	0.0166±0.00228
	Adrenal gl. (L)	0.0116±0.00346	0.0116±0.00397	0.0114±0.00286	0.0122±0.00384	0.0166±0.00234
	Ovary (R)	0.0472±0.01197	0.0440±0.01504	0.0514±0.03374	0.0433±0.01102	0.0381±0.00941
	Ovary (L)	0.0452±0.01522	0.0436±0.01383	0.0432±0.00835	0.0427±0.00962	0.0394±0.00829
	Thymus	0.1057±0.02227	0.0970±0.02412	0.1109±0.02395	0.1097±0.02702	0.1326±0.02355
	Heart	0.5214±0.04078	0.4727±0.05139	0.4772±0.06159	0.4808±0.04046	0.5337±0.03907
	Lung	0.6860±0.12016	0.6466±0.08533	0.6580±0.12504	0.6359±0.07578	0.7358±0.12210
	Brain	1.9093±0.15899	1.7064^a±0.24086^a	1.6069^a±0.17705^a	1.7546±0.13659	2.0028±0.08342
	Pitui gl.	0.0070±0.00270	0.0077±0.00310	0.0084±0.00238	0.0086±0.00203	0.0068±0.00339

All values are expressed as mean±SD, n=10

^a Significantly different from the control group; P<0.05.

Table 6.

Summary incidence of the microscopic observations of mice intradermally treated with human fibroblasts in the 13-week toxicity study

Dosage in (cells/kg B.W.) Tissue/Observation	Male					Female
Dosage in (cells/kg B.W.) Tissue/Observation	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	⁷ 7.5×10⁷ (syngeneic)	0 (Control)	7.5×10⁵	7.5×10⁶	7.5×10⁷	7.5×10⁷ (syngeneic)
Liver
Number examined	10	10	10	10	10	10	10	10	10	10
Glycogen vacuolation	0	1	0	0	0	0	0	0	0	0
Centrilobular vacuolation	0	0	0	0	9	0	0	0	0	0
Parenchymal inflam cell foci	0	0	1	0	0	1	0	0	0	0
Eosinophilic focus	0	0	0	0	0	0	1	0	0	1
Kidneys
Number examined	10	10	10	10	10	10	9	10	10	10
Cortical cyst	1	0	0	0	0	0	0	0	0	0
Pelvic dilatation	1	0	0	0	0	0	0	0	0	0
Cortical vacuolation	1	0	0	0	0	0	0	0	0	0
Nephropathy	1	0	0	0	0	0	0	0	0	0
Subtransi. epi. inflam. infil	3	5	5	3	2	5	7	5	4	4
Interstitial inflam cells	2	4	2	0	1	1	4	1	0	1
Tubular cast(s)	0	2	0	1	0	0	0	0	0	0
Tubular basophilia	0	1	2	1	1	1	0	1	0	0
Tubular hypertrophy, focal	0	0	0	0	0	0	0	0	1	0
Adrenal glands
Number examined	8	10	9	10	10	10	9	10	10	10
Cortical inflammatory cells	0	0	0	1	1	1	1	1	0	0
Pigmentation	0	0	0	0	0	0	0	0	0	1
Subcapsular cell focus	0	0	1	2	8	5	8	5	6	9
Urinary bladder
Number examined	10	10	10	10	10	10	9	10	10	10
Transi epi-subepi inflam cells	0	0	0	0	0	0	1	0	0	0
Spleen
Number examined	10	10	10	10	10	10	10	10	10	10
Haemopoiesis	2	6	5	7	8	8	7	8	6	2
Pancreas
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Thymus
Number examined	2	0	3	4	10	10	10	10	10	9
Involution/atrophy	2	0	3	4	3	6	7	4	7	5
Thyroid
Number examined	5	7	8	8	9	10	7	8	5	9
Ectopic thymus	0	0	0	0	0	1	0	2	3	0
Follicular distension	0	0	0	0	0	0	0	0	1	0
Parathyroid glands
Number examined	1	3	2	1	2	3	0	6	3	1
Lymphoid cells	0	0	0	0	0	1	0	0	0	0
Trachea
Number examined	8	9	10	9	10	9	8	9	6	9
Imflam cell debris in lumen	0	0	0	0	0	0	0	0	0	1
Subepithelial inflam cells	0	0	0	0	1	0	1	0	0	0
Oesophagus
Number examined	9	10	10	10	10	10	8	8	9	10
Submucosal inflammatory cells	0	0	0	0	0	0	0	0	0	1
Tongue
Number examined	10	10	10	10	10	10	10	10	10	10
Mast cells	0	0	1	0	0	0	0	0	0	0
Lung
Number examined	10	10	10	10	10	10	10	10	10	10
Perivascular inflam cells	0	0	1	0	0	1	0	0	0	0
Alveolar macrophages	0	0	1	0	0	0	0	0	0	0
Osseous metaplasia	0	0	0	0	0	0	0	0	0	1
Heart
Number examined	10	10	10	10	10	10	10	10	10	10
Myocardial inflam cells	0	1	0	1	0	0	0	0	0	0
Epicardial inflammatory cells	0	0	0	0	0	0	0	0	0	1
Salivary gland
Number examined	10	10	10	10	10	10	10	10	10	10
Perivascular inflam cells	0	0	0	0	0	0	1	0	0	0
Cervical lymph nodes
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Forestomach
Number examined	10	10	10	10	10	10	10	10	10	10
Submucosal inflammatory cells	0	0	0	0	2	0	0	0	0	0
Epithelial inflam cells	0	0	0	0	1	0	0	0	0	0
Glandular stomach
Number examined	10	10	10	10	10	10	10	10	10	10
Glandular cyst(s)	0	1	0	0	0	0	0	1	0	0
Submucosal inflammatory cells	0	2	3	1	1	0	1	0	0	0
Epithelial hyperplasia	0	0	1	0	0	0	0	0	0	0
Small intestine
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Large intestine
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Mesenteric lymph nodes
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Preputial/Clitoral glands
Number examined	10	10	10	10	10	9	9	9	9	10
Inflammation	1	0	1	2	2	2	1	0	0	0
Skin/mammary gland
Number examined	10	10	10	9	10	10	10	10	10	10
Scab	1	0	0	0	0	0	0	0	0	0
Pannicu muscle-inflam cells	0	0	0	0	1	0	0	0	0	0
Epidermal hyperplasia	1	0	0	0	0	0	0	0	0	0
Deep dermal inflam cells	0	0	0	0	0	0	1	0	0	0
Pannicu muscle-degeneration	0	0	0	0	1	0	0	0	0	0
Subcutaneous granuloma	0	0	0	1	0	0	0	0	1	0
Eye
Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Haderian gland
Number examined	10	10	10	10	10	10	10	10	10	10
Inflammatory cell infiltration	0	0	0	0	0	0	1	0	0	0
Brain
Brain Number examined	10	10	10	10	10	10	10	10	10	10
No abnormalities detected	10	10	10	10	10	10	10	10	10	10
Pituitary
Number examined	8	9	10	9	10	9	10	9	10	10
Number examined Cyst	8 1	9 0	10 0	9 0	10 0	9 0	10 0	9 0	10 0	10 0
Femur/Bone marrow
Number examined	10	10	10	10	10	10	10	10	10	10
Granulocyte hyperplasia	0	0	1	0	0	0	0	0	0	0
Nasal turbinates
Number examined	10	10	10	10	10	10	10	10	10	10
Olf epi-eosinophilic inclusion	4	6	2	3	1	4	7	5	5	5
Inflam cell debris in lumen	1	0	0	0	1	0	1	1	0	0
Olf epi-inflammatory cells	1	0	0	0	0	0	0	1	0	0
Olf epi-subepi inflam cells	1	0	0	0	0	0	0	0	0	0
Resp epi-inflammatory cells	1	3	0	3	0	1	0	0	0	0
Resp epi-subepi inflam cells	1	5	2	3	4	4	3	2	1	2
Resp epi-eosinophilic inclusio	2	3	1	2	1	1	5	3	4	7
Transi epi-inflam cells	0	3	1	3	1	0	0	0	0	0
Transi epi-subepi inflam cells	0	3	0	3	1	1	1	0	0	0
Resp epi-erosin	0	0	0	1	0	0	0	0	0	0
Resp epi-hyperplasia	0	0	0	0	0	0	0	0	1	0
Sternum+marrow
Number examined	10	10	10	10	10	10	10	10	10	10
Granulocyte hyperplasia	0	1	0	0	0	0	0	0	0	0
Sciatic nerve
Number examined	9	10	9	10	9	10	10	10	10	10
Perivascular inflam cells	0	0	0	0	0	1	0	0	0	0
Testis
Number examined	10	10	10	10	10	-	-	-	-	-
Seminiferous tubular degenerat	0	1	0	1	1	-	-	-	-	-
Seminiferous tubular vacuolati	0	0	0	0	1	-	-	-	-	-
Rete tubular hyperplasia	1	0	0	0	0	-	-	-	-	-
Epididymis
Number examined	10	10	10	10	10	-	-	-	-	-
Sperm granuloma	0	0	0	1	0	-	-	-	-	-
Degenerate germ cells in lumen	0	0	0	0	2	-	-	-	-	-
Prostate
Number examined	10	10	10	10	10	-	-	-	-	-
No abnormalities detected	10	10	10	10	10	-	-	-	-	-
Seminal vesicle
Number examined	10	10	10	10	10	-	-	-	-	-
No abnormalities detected	10	10	10	10	10	-	-	-	-	-
Ovary
Number examined	-	-	-	-	-	10	10	10	10	10
Cyst	-	-	-	-	-	0	3	1	2	0
Follicular cyst	-	-	-	-	-	1	0	0	0	0
Prominent corpus leuteum	-	-	-	-	-	0	0	0	2	0
Endometriosis	-	-	-	-	-	0	0	0	0	1
Uterus
Number examined	-	-	-	-	-	10	10	10	10	10
Cystic glands	-	-	-	-	-	2	1	0	0	2
Luminal dilatation	-	-	-	-	-	4	5	6	3	3
Deciduoma	-	-	-	-	-	0	1	0	0	0
Vagina
Number examined	-	-	-	-	-	10	10	10	10	10
Inflam cell debris in lumen	-	-	-	-	-	0	0	0	1	0
Injection site
Number examined	10	10	10	10	10	10	10	10	10	10
Scab	0	0	2	0	0	0	0	0	0	0
Pannicu muscle-regeneration	2	1	5	3	1	0	0	0	0	0
Pannicu muscle-inflam cells	2	1	5	4	1	0	0	0	0	1
Epidermal hyperplasia	0	0	1	0	0	0	0	0	0	0
Deep dermal inflam cells	1	3	2	0	5	2	2	1	1	0
Pannicu muscle fibrosis	0	1	0	1	1	0	0	0	0	0
moderate	0	0	0	1	0	0	0	0	0	0
Pannicu muscle-degeneration	0	0	2	0	0	0	0	0	0	0
Deep derm foreign body granulo	0	0	0	5	2	0	0	0	6	0
Subcutaneous inflam cells	0	0	0	0	1	0	0	0	0	1

TOOLS

Similar articles