Retention of Endogenous Viable Cells Enhances the Anti-Inflammatory Activity of Cryopreserved Amnion

Abstract

Objective: Human amniotic membrane (hAM) has been used to treat wounds for more than 100 years. However, widespread use of fresh hAM has been limited due to its short shelf life and safety concerns. To overcome these concerns, different preservation methods have been introduced. The majority of these methods result in devitalized hAM (dev-hAM). Recently, we developed a cryopreservation method that retains all hAM components intact (int-hAM), including viable endogenous cells. To understand the advantages of retaining viable cells in preserved hAM, we compared the anti-inflammatory properties of int-hAM and dev-hAM. Approach: The tissue composition of int-hAM and dev-hAM was compared with fresh hAM through histology and cell viability analysis. We also evaluated the ability of int-hAM and dev-hAM to regulate tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), and IL-10 release when co-cultured with immune cells; to produce prostaglandin E2 (PGE2) on TNF-α stimulation; and to inhibit proteases. Results: Int-hAM maintained the structural and cellular integrity of fresh hAM. Int-hAM had >80% cell viability post-thaw and remained viable for at least a week in culture. Viable cells were not detected in dev-hAM. Compared with dev-hAM, int-hAM showed significantly greater downregulation of TNF-α and IL-1α, upregulation of PGE2 and IL-10, and stronger inhibition of collagenase. Innovation and Conclusion: A new cryopreservation method has been developed to retain all native components of hAM. For the first time, we show that viable endogenous cells significantly augment the anti-inflammatory activity of cryopreserved hAM.

Figures

Yi Duan-Arnold, PhD

Figure 1.

Hematoxylin and eosin (H&E) and collagen IV (Col IV) staining of human amniotic membranes (hAMs). H&E staining of (A) fresh hAM, (B) viable intact cryopreserved hAM (int-hAM), and (C) devitalized cryopreserved hAM (dev-hAM). Layers of hAM are shown in (A): (i) epithelial layer, (ii) basement membrane, (iii) compact layer, and (iv) stromal layer. Intracytoplasmic vacuoles in the epithelial cells (arrows) are shown in (C). Brown-colored stained Col IV in the basement membrane of (D) fresh-hAM, (E) int-hAM, and (F) dev-hAM. Scale bars: 50 μm (H&E) and 20 μm (Col IV).

Figure 2.

Live and dead endogenous cell staining of human amniotic membrane (hAM). Live and dead cells in epithelial and stromal layers of fresh hAM (A, B), viable intact cryopreserved hAM (int-hAM) post-thaw (C, D), and devitalized cryopreserved hAM (dev-hAM) post-thaw (E, F) were visualized microscopically using the LIVE/DEAD® viability/cytotoxicity kit. Live cells stained green with calcein AM. Dead cells stained red with ethidium homodimer-1. Live and dead cells in int-hAM after 7 days in culture are shown in (G, H). All images were taken at 10× magnification.

Figure 3.

Effects of human amniotic membrane (hAM) on human peripheral blood mononuclear cell (hPBMC)-induced pro-inflammatory factor release. Inhibition of activated hPBMC-derived tumor necrosis factor-α (TNF-α) (A) and interleukin-1α (IL-1α) (B) in the presence of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM). Negative (Neg) and positive (Pos) controls were unstimulated and anti-cluster of differentiation (CD)3 and anti-CD28-activated hPBMCs, respectively. Levels of prostaglandin E2 (PGE2) released from int-hAM and dev-hAM following TNF-α stimulation are shown in (C). PGE2 levels were calculated as % of PGE2 released in cultures without TNF-α (% of control). Data shown represent the mean±SD of one experiment performed in triplicate. Student's t-test was used for statistical analysis. *p<0.05 and ***p<0.001.

Figure 4.

Effects of human amniotic membrane (hAM) on human peripheral blood mononuclear cell (hPBMC)-induced anti-inflammatory factor interleukin-10 (IL-10) release. IL-10 levels in conditioned medium were measured after 24 h co-cultures of lipopolysaccharide (LPS)-stimulated hPBMCs with viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM). Negative (Neg) and positive (Pos) controls were unstimulated and LPS-activated hPBMCs, respectively. Data shown represent the mean±SD of one experiment performed in triplicate. Student's t-test was used for statistical analysis. ***p<0.001.

Figure 5.

Effects of human amniotic membrane (hAM) on protease activities. Substrate cleavage by (A) Collagenase Type IV and (B) neutrophil elastase in the presence of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM) was measured spectrophotometrically and presented in absorbance. Proteases hydrolyze substrates and yield soluble colored peptides in proportion to enzyme activity. Collagenase Type IV activity was assayed using azocoll as a substrate. Elastase activity was assayed using methoxysuccinyl-ala-ala-pro-val-p-nitroanilide as a substrate. Negative (Neg) and positive (Pos) controls were substrates alone and substrates incubated with proteases, respectively. Data shown represent the mean±SD of one experiment performed in triplicate. Student's t-test was used for statistical analysis.*p<0.05 and ***p<0.001.

Figure 6.

Summary of viable intact cryopreserved human amniotic membrane (int-hAM) and devitalized cryopreserved human amniotic membrane (dev-hAM) properties. The magnitude of effect between int-hAM and dev-hAM was calculated using the following formula: ((% change of int-hAM)/(% change of dev-hAM))*100. In instances where the % change was negative, magnitude of effect of int-hAM relative to dev-hAM, expressed as % of dev-hAM, was calculated using the following formula: ([(% change of int-hAM)−(% change of dev-hAM)]/[absolute value of (% change of dev-hAM)])*100.