Application of 3D skin culture model as a tool to study the role of immune mechanisms in chronic diabetic foot ulcers pathogenesis

Maryia Mashkova; Vitaly Goranov; Tatiana Mokhort; Alena Shyshko; Marina Mantachik

doi:10.1530/endoabs.63.GP33

The aim: To test cytotoxic effects of immune factors of patients with chronic diabetic foot ulcers on keratinocytes and fibroblasts in a 3D skin culture system.

Materials and methods: In this study a multilayer 3D human skin model comprising of keratinocytes, fibroblasts and dendritic cells in an agarose-fibronectin gel was used. 20% serum of 13 patients with chronic noninfected diabetic foot ulcers, 13 diabetic type 2 patients and 13 healthy people and lymphocyte/monocyte mixture of the same groups of patients were added to the culture model. The system was also tested with standard irritants – dimethyl sulfoxide, Lipopolysaccharide. Cell viability and growth of fibroblasts and keratinocytes was measured using the resazurin reduction assay.

Results: Decreased fibroblasts viability was seen in the presence of blood components of patients with chronic diabetic foot ulcers (blood serum and lymphocyte/monocyte mixture). At the same time, the cytoinhibitory effect was observed mainly in the presence of lymphocyte/monocyte mixture of these patients (Table 1).

Table 1 Results of 3D culture model testing.
	Control (standard irritants only, no serum+lymph/monocyte)		Healthy people (20% serum+lymph/monocyte)
Irritant	Keratinocytes (%)	Fibroblasts (%)	Keratinocytes (% to control)	Fibroblasts (% to control)
Control	100	100	-	-
DMSO, (100 mM)	101.1 [98.7;103.5]	104.8 [101.1;108.2]	103.0 [98.7;107.1]	99.8 [98.3;101.1]
SCS, (5 μg/ml)	94.4 [90.1;99.2]	97.1 [93.6;100.3]	103.8 [102.9;104.2]	99.8 [99.1;103.1]
LPS, (10 μg/ml)	108.0 [103.1;112.6]	97.1 [92.1;101.3]	95.5 [92.1;99.2]	76.8 [69.7;87.2]
DMSO+LPS	87.6 [83.3;91.2]	105.1 [102.1;108.6]	84.0 [79.1;89.1]	90.8 [82.3;96.8]
Only 20% serum	-	-	104 [96.7;110.3]	98.3 [96.9;99.7]
Only lymph/monocyte	-	-

Table 1 Continued.
	Diabetic patients (20%serum+lymph/monocyte)		Diabetic foot ulcers patients (20%serum+lymph/monocyte)
Irritant	Keratinocytes (% to control)	Fibroblasts (% to control)	Keratinocytes (% to control)	Fibroblasts (% to control)
Control	-	-	-	-
DMSO, (100 mM)	96.8 [90.4;96.7]	89.6 [82.4;93.1]	92.6 [90.3;95.3]	76.8* [72.3;81.3]
SCS, (5 μg/ml)	92.5 [85.7;99.8]	84.1 [79.3;88.1]	88.7 [85.2;101.1]	70.0* [64.3;78.1]
LPS, (10 μg/ml)	91.7 [76.3;92.4]	77.2 [67.1;82.3]	71.2** [64.8;75.9]	67.8* [62.5;70.6]
DMSO+LPS	86.1 [78.4;92.6]	82.6 [77.4;91.2]	84.2 [76.4;91.2]	66.1* [61.4;69.6]
Only 20% serum	98.8 [86.7;100.2]	95.8 [87.2;101.2]	94.7 [84.8;99.6]	93.2 [88.1;96.2]
Only lymph/monocyte	93.6 [85.8;98.1]	89.1 [74.2;95.3]	87.1** [83.2;91.4]	64.7* [59.8;67.9]
**Significant difference vs healthy group and control, P<0.05.
*Significant difference vs diabetic, healthy group and control, P<0.05;

Conclusion: 3D skin culture model can be used to study in vitro the role of immune factors in pathogenesis of chronic diabetic foot ulcers.

Endocrine Abstracts

GP33