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

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

Affiliations
  • 1Department of Experimental Animal Research, Clinical Research Institute, Seoul National University Hospital, Seoul, Korea. bckang@snu.ac.kr
  • 2Department of Immunology and Laboratory Animal Medicine, College of Medicine, Seoul National University, Seoul, Korea.
  • 3S.BIOMEDICS Co., Ltd. Seoul, Korea.

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 7.5x10(7) 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.

Keyword

Fibroblast; tumorigenicity; toxicity; distribution; cell therapy

MeSH Terms

Animals
Biochemistry
Body Weight
Carcinogenicity Tests
Cicatrix
Dermis
Fibroblasts
Fluorescence
Hematology
Humans
Mice
Mice, Nude
No-Observed-Adverse-Effect Level
Retention (Psychology)
Tissue Therapy
Transplants

Figure

  • 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).


Reference

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