Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Quasi-natural cell block; Transplantation; Scaffold; Extracellular matrix; Hydrogel

Authorship

H.J. Chung

Department of Biomedical Sciences, Institute for Medical Science, Chonbuk National University Medical School, Jeonju, Chonbuk, South KoreaChonbuk National University Medical SchoolSouth KoreaJeonju, Chonbuk, South KoreaDepartment of Biomedical Sciences, Institute for Medical Science, Chonbuk National University Medical School, Jeonju, Chonbuk, South Korea

* These authors contributed equally to this study.

M.M. Hassan

* These authors contributed equally to this study.

J.O. Park

H.J. Kim

JINIS BDRD Institute, JINIS Biopharmaceuticals Co., Wanju, Chonbuk, South KoreaJINIS Biopharmaceuticals Co.South KoreaWanju, Chonbuk, South KoreaJINIS BDRD Institute, JINIS Biopharmaceuticals Co., Wanju, Chonbuk, South Korea

S.T. Hong

Correspondence: Seong-Tshool Hong: <seonghong@chonbuk.ac.kr>

*
These authors contributed equally to this study.

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Figures

Figures (6)

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Figure 1
Schematic diagram of strategies to transplant adherent somatic cells. A, Transplantation of adherent somatic cells based on a mixture approach. B, Transplantation of adherent somatic cells by 3D culture. C, Transplantation of adherent somatic cells through manipulation of the quasi-natural cell block.

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Figure 2
Preparation of adipose-derived mesenchymal stromal (ADMS) cells to manipulate the quasi-natural cell block. ADMS cells were isolated from peritoneal adipose tissue excised from the abdominal region of male C57BL/6 mice and cultured for in vitro expansion. Mouse ADMS cells at passage 2 (A) and passage 6 (B) showed similar fibroblast-like morphology. The growth curve of ADMS cells was determined at passage 6 by using the MTT assay (C); an initial lag phase of 2 days, a log phase from day 3 to day 5, and a plateau phase.

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Figure 3
In vitro construction and implantation of the quasi-natural cell block. The quasi-natural cell block cultured for 7 days (A) was harvested by removing its surrounding Matrigel™-cast for transplantation (B). The quasi-natural cell block was implanted into subcutaneous tissue of a female mouse (C) and allowed to adapt in vivo for 4 weeks, showing successful connection with the surrounding tissue of the recipient (D).

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Figure 4
Evaluation of the quasi-natural cell block. ADMS cells were mixed with Matrigel™ gel to transplant the mixture in a conventional approach and tissue sections were prepared for histochemical staining (A, B). The quasi-natural cell block (C, D) was manipulated and histologically evaluated in comparison with the conventional method. In the conventional method, H&E staining showed mostly scaffold materials with a few encapsulated cells in the mixture before transplantation (A) and after transplantation (B). In the quasi-natural cell block, however, much higher densities of ADMS cells were observed before transplantation (C) as well as after transplantation (D).

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Figure 5
Successful regeneration of the quasi-natural cell block in vivo. H&E staining showed small numbers of the transplanted ADMS cells in the conventional mixture approach as well as a general unhealthy shape of the transplanted cells (A-C). In contrast, the quasi-natural cell block implants displayed successful proliferation of implanted ADMS cells (D, E). Morphological examinations further revealed differentiation of ADMS cells into various cell types as well as formation of capillary vessels with erythrocytes (E-I). Arrows indicate erythrocytes (E, G), adipocytes (F), or multinuclear giant cells (H, I).

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Figure 6
Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus (A, B). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue (C) along with native heart tissue (D). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated (E). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin (F). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: (A, C, D, E and F) 20 μm; (B) 100 μm.

Figure 1 Schematic diagram of strategies to transplant adherent somatic cells. A, Transplantation of adherent somatic cells based on a mixture approach. B, Transplantation of adherent somatic cells by 3D culture. C, Transplantation of adherent somatic cells through manipulation of the quasi-natural cell block.

Figure 2 Preparation of adipose-derived mesenchymal stromal (ADMS) cells to manipulate the quasi-natural cell block. ADMS cells were isolated from peritoneal adipose tissue excised from the abdominal region of male C57BL/6 mice and cultured for in vitro expansion. Mouse ADMS cells at passage 2 (A) and passage 6 (B) showed similar fibroblast-like morphology. The growth curve of ADMS cells was determined at passage 6 by using the MTT assay (C); an initial lag phase of 2 days, a log phase from day 3 to day 5, and a plateau phase.

Figure 3 In vitro construction and implantation of the quasi-natural cell block. The quasi-natural cell block cultured for 7 days (A) was harvested by removing its surrounding Matrigel™-cast for transplantation (B). The quasi-natural cell block was implanted into subcutaneous tissue of a female mouse (C) and allowed to adapt in vivo for 4 weeks, showing successful connection with the surrounding tissue of the recipient (D).

Figure 4 Evaluation of the quasi-natural cell block. ADMS cells were mixed with Matrigel™ gel to transplant the mixture in a conventional approach and tissue sections were prepared for histochemical staining (A, B). The quasi-natural cell block (C, D) was manipulated and histologically evaluated in comparison with the conventional method. In the conventional method, H&E staining showed mostly scaffold materials with a few encapsulated cells in the mixture before transplantation (A) and after transplantation (B). In the quasi-natural cell block, however, much higher densities of ADMS cells were observed before transplantation (C) as well as after transplantation (D).

Figure 5 Successful regeneration of the quasi-natural cell block in vivo. H&E staining showed small numbers of the transplanted ADMS cells in the conventional mixture approach as well as a general unhealthy shape of the transplanted cells (A-C). In contrast, the quasi-natural cell block implants displayed successful proliferation of implanted ADMS cells (D, E). Morphological examinations further revealed differentiation of ADMS cells into various cell types as well as formation of capillary vessels with erythrocytes (E-I). Arrows indicate erythrocytes (E, G), adipocytes (F), or multinuclear giant cells (H, I).

Figure 6 Confirmation of the allografted quasi-natural cell block in the recipient body. To confirm allogenicity of the transplanted quasi-natural cell block, gender-specific probes were used to distinguish the male-derived allografted tissue from the recipient female mouse by FISH along with DAPI staining of the nucleus (A, B). Green spots indicate male Y chromosome probe in the corresponding female tissue. For intercellular communication capability in the allografted tissue, specimens were stained with specific probes for signaling marker connexin43, in the quasi-natural tissue (C) along with native heart tissue (D). Immunostaining of the allografted quasi-natural tissue with epithelial linage marker CD31 revealed vascular network assembly as indicated (E). Immunostaining of the allografted quasi-natural tissue further showed that most of cells were stained with anti-vimentin, mesodermal lineage marker, but not with anti-cytokeratin (F). Images for individual channels (connexin43 with alexa 488 is green, cd31 with alexa 568 is red, vimentin with alexa 488 is green, cytokeratin with alexa 568 is red) are shown on the left, and main panels show the merged image containing all channels plus DIC. The cells nuclei were visualized with DAPI (blue). Scale bar: (A, C, D, E and F) 20 μm; (B) 100 μm.

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Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

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[1] Correspondence: Seong-Tshool Hong: <seonghong@chonbuk.ac.kr>