BackgroundMesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity.MethodsPlatelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α.ResultsOur results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed the release of TNF-α when exposed to LPS-stimulated monocytes similar to those cultured in FBS.ConclusionePL has the potential to be used for the expansion of MSCs before clinical application, avoiding the concerns associated with the use of FBS.

The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.

There is a clear need for low-cost, self-applied, long-lasting approaches to prevent human immunodeficiency virus (HIV) infection in both men and women, even with the advent of pre-exposure prophylaxis (PrEP). Broadly neutralizing antibodies represent an option to improve HIV prophylaxis, but intravenous delivery, cold-chain stability requirements, low cervicovaginal concentrations, and cost may preclude their use. Here, we present an approach to express the anti-GP120 broadly neutralizing antibody PGT121 in the primary site of inoculation, the female reproductive tract, using synthetic mRNA. Expression is achieved through aerosol delivery of unformulated mRNA in water. We demonstrated high levels of antibody expression for over 28 days with a single mRNA administration in the reproductive tract of sheep. In rhesus macaques, neutralizing antibody titers in secretions developed within 4 h and simian-HIV (SHIV) infection of ex vivo explants was prevented. Persistence of PGT121 in vaginal secretions and epithelium was achieved through the incorporation of a glycosylphosphatidylinositol (GPI) anchor into the heavy chain of the antibody. Overall, we present a new paradigm to deliver neutralizing antibodies to the female reproductive tract for the prevention of HIV infections.

March 2021Commonly misidentified lines (See ICLAC register) Neither Expi293F nor A549 cells are listed as misidentified cell lines in the ICLAC register.

Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture.

IntroductionExtracorporeal shockwave therapy (ESWT) and platelet-rich plasma (PRP) are common treatments for soft tissue injuries in horses. Shockwave triggers cell specific responses to promote healing. Growth factors released from PRP also promote healing. It has been hypothesized that greater growth factor release would amplify the healing process. The combination of ESWT and PRP could promote healing in injured tendons and ligaments in the horse. The objective of this study was to determine if application of shockwaves to PRP samples increases the concentration of transforming growth factor-β1 (TGF-β1) and platelet-derived growth factor ββ (PDGF-ββ) released from the platelets in vitro.Materials and methodsPRP was produced from blood drawn from six horses. The PRP from each horse was exposed to the following treatments: (1) positive control (freeze-thaw cycle), (2) untreated negative control, or shockwaves with either (3) a “standard probe” (ESWT-S) with a 2 cm focal width and medium energy density or (4) a “power probe” (ESWT-P) with a 1 cm focal width and high energy density. After each treatment, the samples were centrifuged, and the supernatant was harvested. The supernatant was then used for growth factor quantification via commercially available ELISA kits for TGF-β1 and PDGF-ββ.ResultsConcentrations of TGF-β1 and PDGF-ββ in PRP that underwent a freeze-thaw cycle were significantly increased compared with all other treatments. Both ESWT-S and ESWT-P resulted in significantly increased TGF-β1 concentrations, 46 and 33%, respectively, when compared with the negative control. Both ESWT-S and ESWT-P resulted in significantly increased PDGF-ββ concentrations, 219 and 190%, respectively, when compared with the negative control.DiscussionThese data indicate that the application of ESWT to PRP increases the expression of growth factors in vitro. This suggests that the combination therapy of local PRP injection followed by ESWT may stimulate release of growth factors from platelets after they have been injected into the area of injury.ConclusionThe combination of PRP and ESWT might result in synergism of two modalities previously utilized individually for tendon and ligament injuries in horses.

Bovine respiratory disease (BRD) linked with Mannheimia haemolytica is the principal cause of pneumonia in cattle. Diagnosis of BRD traditionally relies on visual assessment, which can be untimely, insensitive, and nonspecific leading to inadequate treatment and further spread of disease. Near Infrared Spectroscopy (NIRS) is a rapid acquisition vibrational spectroscopy that can profile changes in biofluids, and when used in combination with multivariate analysis, has potential for disease diagnosis. This study characterizes the NIR spectral profile of blood plasma from dairy calves infected with M. haemolytica and validates the spectral biochemistry using standardized clinical and hematological reference parameters. Blood samples were collected for four days prior to (baseline), and 23 days after, a controlled intrabronchial challenge. NIR spectral profiles of blood plasma discriminated and predicted Baseline and Infected states of animal disease progression with accuracy, sensitivity, and specificity ≥ 90% using PCA–LDA models. These results show that physiological and biochemical changes occurring in the bloodstream of dairy calves during M. haemolytica infection are reflected in the NIR spectral profiles, demonstrating the potential of NIRS as a diagnostic and monitoring tool of BRD over time.

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