Antimicrobial Effects of Conditioned Medium From Amniotic Progenitor Cells in vitro and in vivo: Toward Tissue Regenerative Therapies for Bovine Mastitis

Abstract

There is increasing evidence to suggest that, in addition to their regenerative effect, mesenchymal stromal cells (MSCs), and their secretome have an anti-inflammatory and antimicrobial role in the innate immune response in conditions such as sepsis. However, there is no published information on the effect of MSCs in bovine mastitis. Mastitis often results in extensive tissue damage due to multi-microorganism co-infection. This study investigated the ability of amniotic-derived conditioned medium (CM), in vitro and in vivo, to counteract microbial action and restore healthy tissue capable of milk production. Following determination of a dose-response curve, 10,000 colony-forming units (CFU) of Staphylococcus aureus (S. aureus) were inoculated into bovine mammary epithelial cell culture with and without 10% CM (supplemented either at the time of bacteria inoculation or after 4 h). Acridine orange staining was used to assess cell viability/apoptosis. Additionally, an in vivo study was performed using 48 dairy cows with acute and chronic mastitis, treated with CM (treated group) or antibiotics (control group). In vitro results showed that CM can attenuate bacterial growth, as evaluated by the number of CFU. After 24 h of culture with S. aureus, 89.67% of mammary epithelial cells treated with CM were still alive, whereas all cells cultured without CM were dead. Rates of epithelial cell survival (60.67%) were similar when CM was added 4 h after bacteria inoculation. There was no difference in somatic cell count between cases of acute mastitis in the CM-treated or control group in the in vivo study. However, relapses in chronic mastitis were less common in the group receiving CM. Our results show that CM is able to mitigate bacterial growth in vitro and may be particularly useful in the treatment of chronic mastitis, aiding restoration of milk production in cows that would otherwise be removed from the production cycle.

Microfluidic Tools for Enhanced Characterization of Therapeutic Stem Cells and Prediction of Their Potential Antimicrobial Secretome

“…Encouraging reports have been recorded in the veterinary field, mainly in the treatment of mastitis models using CM from bovine bone marrow and adipose tissue MSCs [97]. In another study, CM treatment led to a lower rate of relapses than with antibiotic treatment [98].…”

Mesenchymal Stromal Cells as Potential Antimicrobial for Veterinary Use—A Comprehensive Review

“…Ease of isolation was likely a factor in choosing BM or AT for some (Johnson et al, 2017;Cortés-Araya et al, 2018;Cahuascanco et al, 2019;Bujňáková et al, 2020;Peralta et al, 2020) and peripheral blood (PB) for others (Harman et al, 2017;Marx et al, 2020). Still, potency and lack of immunogenicity were also cited as reasons for choosing certain tissue sources, which led more than one group to isolate MSCs from fetal sources of BM, AT, or the amniotic membrane (Cahuascanco et al, 2019;Lange-Consiglio et al, 2019;Peralta et al, 2020). Although fetal and perinatal tissue-derived MSCs have been reported to be superior to their adult-derived counterparts in their proliferative potential and hypoimmunogenicity (Deus et al, 2020), it is as yet unclear what effect, if any, their more primitive state might have on their antimicrobial activity.…”

“…Consequently, the secretome can be influenced by culture conditions that activate or precondition MSCs to respond to the clinical problem at hand as discussed above. In the articles reviewed here, only two of the studies took strictly a cellular approach (Johnson et al, 2017;Peralta et al, 2020), one assessed both cells and CM (Harman et al, 2017), while the rest worked only with CM (Cortés-Araya et al, 2018;Cahuascanco et al, 2019;Lange-Consiglio et al, 2019;Bujňáková et al, 2020;Marx et al, 2020). Co-culturing equine PB-MSCs with either E. coli or S. aureus, Harman et al compared direct contact with transwell separation and found that both the cells and the paracrine factors alone had an inhibitory effect albeit somewhat muted in the case of the transwell setup (Harman et al, 2017).…”

“…In bovine studies, two recent independent articles examining MSC therapies for mastitis in dairy cows indicate that MSCs may become a useful tool for production animals as well (Lange-Consiglio et al, 2019;Peralta et al, 2020). In the first study, fourfold concentrated CM reconstituted from lyophilized allogeneic non-primed bovine amniotic tissue-derived MSC CM were used for both in vitro and in vivo studies (Lange-Consiglio et al, 2019). The in vivo study enrolled 48 dairy cows with either acute or chronic mastitis and compared CM versus antibiotic treatment.…”

“…The in vitro study had S. aureus-inoculated bovine mammary epithelial cell cultures set up with 10% MSC CM added at the time of inoculation, 4 h later, or not at all. After 24 h, epithelial cell survival rates were 90% for cultures when supplemented by CM at time 0, 61% when supplemented at 4 h, and 0% when no CM was added (Lange-Consiglio et al, 2019). In the second study, S. aureus mastitis was experimentally induced in 15 Holstein Friesian cows who were then treated intramammarily twice within 10 days with either allogeneic non-primed bovine AT-MSCs (2.5×10 7 /dose), antibiotics, or ringer lactate (negative control; vehicle) (Peralta et al, 2020).…”