Table 3 Methods for evaluating in vitro biocompatibility and cell proliferative activity of hydrogels loaded with cell conditioned medium using model cell lines

Wound scratch assay

Human umbilical vein endothelial cells

The migration rate is measured as the difference of the width in between cell monolayer parts at predetermined time points

Enhanced cell migration and faster wound closure

[60]

Human skin fibroblasts

[54]

Fibroblasts or human umbilical vein endothelial cells

[69]

Human dermal fibroblast

[58]

Immortalized human HaCaT keratinocytes

[70]

[52]

Cell proliferation assay

Human mesenchymal stromal cells

Cell Counting Kit-8 assay

Good biocompatibility, low cytotoxicity, clearly supported cell survival and proliferation

[64]

Human umbilical vein endothelial cells

[63]

RAW 264.7 murine-derived macrophages

[57]

Human fibroblasts and human umbilical vein endothelial cells

[69]

L929 murine fibroblasts

[68]

Collagen deposition assay

Human skin fibroblasts

Quantitative analysis of immunofluorescence staining collagen type I

Promoted collagen synthesis

[54]

Human dermal fibroblasts

[58]

Tube formation assay

Human umbilical vein endothelial cells

Quantitative microscopic analysis of loop number

Promoted tube formation

[60]

[63]

Cell migration assay

Human epidermal CB-HK-001 keratinocytes

Transwell membrane assay (8 mm pore size)

Induced cell migration

[54]

Human umbilical vein endothelial cells

[63]

Phenotype change analysis

Murine bone marrow macrophages

Quantitative real-time PCR of total RNA (GAPDH, CD206, CCR7 genes)

Upregulated expression of the M2 macrophage marker CD206, reduced expression of the M1 macrophage marker CCR7

[63]

RAW 264.7 murine-derived macrophages

Expression of macrophage markers, including induced nitric oxide synthase (iNOS, M1), and arginase (ARG, M2) by flow cytometry

Stimulation of M2-polarization of macrophages

[57]

Fibroblast differentiation assay

Human fibroblasts

α-SMA mRNA expression

Downregulation of α-SMA expression; inhibition of proliferation fibroblasts into myofibroblasts leading to scarless healing

[69]

Inflammation gene expression

RAW 264.7 murine-derived macrophages

Quantitative real-time PCR of total RNA

Supression of pro-inflammation gene IL-23 expression and upregulation of anti-inflammation gene IL-10

[57]

Immune response

Human peripheral blood mononuclear cells

Cell proliferative response due to incorporation of tritiated thymidine (3 H-TdR) and its DNA binding is analyzed

No significant difference in PBMC proliferation (no immune response)

[61]

Cell viability

Human dermal fibroblasts

MTT assay

No cytotoxic effect, increased cell proliferation

[55]

Assessment of cytoprotectivity against H₂O₂ toxicity

Human mesenchymal stromal cells

MTT assay, additional treatments of the cells with 250 µM H₂O₂ solution for 24 h to induce cell death

Significant reduction in cell death caused by H₂O₂

[65]