In Vivo Longitudinal Monitoring of Cardiac Remodeling in Murine Ischemia Models With Adaptive Bayesian Regularized Cardiac Strain Imaging: Validation Against Histology.

Adaptive Bayesian regularized cardiac strain imaging (ABR-CSI) uses raw radiofrequency signals to estimate myocardial wall contractility as a surrogate measure of relative tissue elasticity incorporating regularization in the Bayesian sense. We determined the feasibility of using ABR-CSI -derived strain for in vivo longitudinal monitoring of cardiac remodeling in a murine ischemic injury model (myocardial infarction [MI] and ischemia-reperfusion [IR]) and validated the findings against ground truth histology. We randomly stratified 30 BALB/CJ mice (17 females, 13 males, median age = 10 wk) into three surgical groups (MI = 10, IR = 12, sham = 8) and imaged pre-surgery (baseline) and 1, 2, 7 and 14 d post-surgery using a pre-clinical high-frequency ultrasound system (VisualSonics Vevo 2100). We then used ABR-CSI to estimate end-systolic and peak radial (er) and longitudinal (el) strain estimates. ABR-CSI was found to have the ability to serially monitor non-uniform cardiac remodeling associated with murine MI and IR non-invasively through temporal variation of strain estimates post-surgery. Furthermore, radial end-systole (ES) strain images and segmental strain curves exhibited improved discrimination among infarct, border and remote regions around the myocardium compared with longitudinal strain results. For example, the MI group had significantly lower (Friedman's with Bonferroni-Dunn test, p = 0.002) ES er values in the anterior middle (infarcted) region at day 14 (n = 9, 9.23 ± 7.39%) compared with the BL group (n = 9, 44.32 ± 5.49). In contrast, anterior basal (remote region) mean ES er values did not differ significantly (non-significant Friedman's test, χ2 = 8.93, p = 0.06) at day 14 (n = 6, 33.05 ± 6.99%) compared with baseline (n = 6, 34.02 ± 6.75%). Histology slides stained with Masson's trichrome (MT) together with a machine learning model (random forest classifier) were used to derive the ground truth cardiac fibrosis parameter termed histology percentage of myocardial fibrosis (PMF). Both radial and longitudinal strain were found to have strong statistically significant correlations with the PMF parameter. However, radial strain had a higher Spearman's correlation value (εresρ = -0.67, n = 172, p < 0.001) compared with longitudinal strain (εlesρ = -0.60, n = 172, p < 0.001). Overall, the results of this study indicate that ABR-CSI can reliably perform non-invasive detection of infarcted and remote myocardium in small animal studies.

Murine cardiac fibrosis localization using adaptive Bayesian cardiac strain imaging in vivo.

An adaptive Bayesian regularized cardiac strain imaging (ABR-CSI) algorithm for in vivo murine myocardial function assessment is presented. We report on 31 BALB/CJ mice (n = 17 females, n = 14 males), randomly stratified into three surgical groups: myocardial infarction (MI, n = 10), ischemia-reperfusion (IR, n = 13) and control (sham, n = 8) imaged pre-surgery (baseline- BL), and 1, 2, 7 and 14 days post-surgery using a high frequency ultrasound imaging system (Vevo 2100). End-systole (ES) radial and longitudinal strain images were used to generate cardiac fibrosis maps using binary thresholding. Percentage fibrotic myocardium (PFM) computed from regional fibrosis maps demonstrated statistically significant differences post-surgery in scar regions. For example, the MI group had significantly higher PFMRadial (%) values in the anterior mid region (p = 0.006) at Day 14 (n = 8, 42.30 ± 14.57) compared to BL (n = 12, 1.32 ± 0.85). A random forest classifier automatically detected fibrotic regions from ground truth Masson's trichrome stained histopathology whole slide images. Both PFMRadial (r = 0.70) and PFMLongitudinal (r = 0.60) results demonstrated strong, positive correlation with PFMHistopathology (p < 0.001).

Age-related effects on the potency of human adipose-derived stem cells: creation and evaluation of superlots and implications for musculoskeletal tissue engineering applications.

Human adipose-derived stem cells (hASC) are now a prevalent source of adult stem cells for studies in tissue engineering and regenerative medicine. However, researchers utilizing hASC in their investigations often encounter high levels of donor-to-donor variability in hASC differentiation potential. Because of this, conducting studies with this primary cell type can require extensive resources to generate statistically significant data. We present a method to generate pooled donor cell populations, termed "superlots," containing cell populations derived from four to five age-clustered donors. The goal of generating these superlots was to 1) increase experimental throughput, 2) to utilize assay resources more efficiently, and 3) to begin to establish global hASC differentiation behaviors that may be associated with donor age. With our superlot approach, we have validated that pooled donor cell populations exhibit proliferative activity representing the combined behavior of each individual donor cell line. Further, the superlots also exhibit differentiation levels roughly approximating the average combined differentiation levels of each individual donor cell line. We established that high donor-to-donor variability exists between the pre-, peri-, and postmenopausal age groupings and that proliferation and differentiation characteristics can vary widely, independent of age. Interestingly, we did observe that cell lines derived from postmenopausal donors demonstrated a relatively high proclivity for osteogenic differentiation and a relatively lowered proclivity for adipogenic differentiation as compared with cells derived from pre- and perimenopausal donors. In general, superlots effectively represented the average differentiation behavior of each of their contributing cell populations and could provide a powerful tool for increasing experimental throughput to more efficiently utilize resources when studying hASC differentiation.

Rapid, large-scale formation of porcine hepatocyte spheroids in a novel spheroid reservoir bioartificial liver.

We have developed a novel bioreactor based on the observation that isolated porcine hepatocytes rapidly and spontaneously aggregate into spheroids under oscillation conditions. The purpose of this study was to characterize the influence of oscillation frequency (0.125 Hz, 0.25 Hz), cell density (1-10 x 10(6) cells/mL), and storage condition (fresh, cryopreserved) of porcine hepatocytes on the kinetics of spheroid formation. The viability and metabolic performance of spheroid hepatocytes was also compared to monolayer culture. We observed that both fresh and cryopreserved porcine hepatocytes began formation of spheroids spontaneously at the onset of oscillation culture. Spheroid size was directly related to cell density and time in culture, though inversely related to oscillatory frequency. Spheroid formation by fresh porcine hepatocytes was associated with decreased cell death (lactate dehydrogenase release, 1.3 +/- 1.0 vs. 3.1 +/- 0.7 U/mL, P < 0.05) and increased metabolic performance (albumin production, 14.7 +/- 3.3 vs. 4.6 +/- 1.4 fg/c/h, P < 0.0001; ureagenesis from ammonia, 267 +/- 63 vs. 92 +/- 13 micromol/L/h, P < 0.001) compared with monolayer culture. In conclusion, based on the favorable properties of rapid spheroid formation, increased hepatocellular function, and ease of scale-up, the spheroid reservoir bioreactor warrants further investigation as a bioartificial liver for support of liver failure.

Cytotoxic immune response to a xenogeneic bioartificial liver.

Prior studies have suggested the possibility of immune-mediated death of xenogeneic hepatocytes in a bioartificial liver (BAL) during hemoperfusion. This study was designed to elucidate how immunity may cause death of xenogeneic hepatocytes in the BAL. Healthy dogs were treated with a BAL containing hollow fiber membranes with large pores (200 nm) or small pores (400 kDa). The immune response of recipient dogs to BAL therapy was monitored over 3 h of treatment. We observed significantly greater loss of viability of hepatocytes in the 200 nm group compared with the 400 kDa group (p < 0.001). Low viability after treatment with the large pore membrane was associated with positive staining for dog IgG, dog IgM, and dog complement on dead hepatocytes. Significant levels of dog antibody were detected in samples of BAL medium from the 200 nm group. These canine antibodies were cytotoxic to porcine hepatocytes. In contrast, medium from the 400 kDa group contained only trace levels of dog IgG and were noncytotoxic. We conclude that antibody-mediated cytotoxicity contributed to the death of hepatocytes during treatment with a xenogeneic BAL. Immune-mediated death of hepatocytes was reduced by increasing selectivity of the BAL membrane.

Membrane pore size impacts performance of a xenogeneic bioartificial liver.

BACKGROUND: We have developed a novel bioartificial liver (BAL) composed of porcine hepatocyte spheroids in a reservoir design. A semipermeable membrane is used to protect the spheroids from immune-mediated damage. This study was designed to assess the influence of membrane pore size on performance of the spheroid reservoir BAL. METHODS: Eight healthy dogs were studied during primary and secondary exposures to the spheroid reservoir BAL using membranes with small (10 nm) or large (200 nm) pores. BAL performance was assessed by multiple functional assays. Spheroids were examined microscopically before and after all BAL treatments. Titers of xenoreactive antibody were monitored until elective death of animals on day 42. RESULTS: Viability and functional performance of spheroids were significantly greater after all BAL treatments that used membranes with 10-nm versus 200-nm pores. Reduced performance in the 200 nm group was associated with 7.7-fold and 78.0-fold rise in xenoreactive antibody titers after first and second treatments, respectively. Dogs in the 10 nm group remained hemodynamically stable during all BAL treatments, whereas those in the 200 nm group experienced acute hypotension (P<0.001) during second BAL exposures. Microscopic examination of spheroids after BAL treatments indicated that deposition of canine proteins, including complement, was associated with reductions in both viability and functional performance of the BAL. CONCLUSIONS: The elicited immune response of healthy dogs to a xenogeneic BAL was blocked and BAL performance significantly improved by reducing the permeability of the BAL membrane.

Apoptotic cell death and function of cryopreserved porcine hepatocytes in a bioartificial liver.

We have previously shown that cryopreservation leads to increased apoptotic death of porcine hepatocytes intended for use in a bioartificial liver (BAL). This study was designed to determine if a broad-spectrum caspase inhibitor, IDN-1965, reduced apoptosis and increased function of cryopreserved porcine hepatocytes in static culture or in a BAL. Porcine hepatocytes were studied immediately after isolation and after 2 weeks of cryopreservation in liquid nitrogen using medium supplemented with 25 micromol/L IDN-1965 or vehicle. Both apoptotic and necrotic cells were observed in cultures of fresh and cryopreserved hepatocytes, but the percentage of apoptotic cells increased after cryopreservation. Cryopreservation in IDN-1965 improved hepatocyte viability and reduced apoptotic cell death determined by TUNEL assay. Cryopreservation of hepatocytes in IDN-1965 was also associated with reduced caspase 3-like activity, decreased release of cytochrome c from mitochondria, and a slower decline in mitochondrial membrane potential after thawing. These markers of apoptosis were lowest after cryopreservation when IDN-1965 was added to both the culture and cryopreservation medium. Functional markers of hepatocyte activity (albumin production, diazepam metabolism, urea production) were also increased after cryopreservation and culture of hepatocytes in medium supplemented with 25 micromol/L IDN-1965. Cryopreservation of porcine hepatocytes in the presence of caspase inhibitor IDN-1965 was associated with reduced apoptosis and improved function of porcine hepatocytes in both static culture and a perfused BAL. These data demonstrate that inhibition of apoptosis also preserves cell function.

Membrane barrier of a porcine hepatocyte bioartificial liver.

Pores in the membrane of a bioartificial liver (BAL) allow it to function as a semipermeable barrier between its contents (i.e., liver cells) and components of the recipient's immune system. This study is designed to assess the influence of pore size on immune response to a BAL containing porcine hepatocytes. Sixteen healthy dogs were divided into four groups (four dogs per group) based on pore size of the BAL membrane and level of exposure to porcine hepatocytes. Group 1 dogs were administered porcine hepatocytes by intraperitoneal injection and served as positive controls. Group 2 dogs were exposed to porcine hepatocytes in a large-pore (200-nm) BAL, and group 3 dogs were exposed to porcine hepatocytes in a small-pore (10-nm) BAL. Group 4 dogs were exposed to a no-cell (unloaded) BAL and served as negative controls. Intraperitoneal injection of hepatocytes or 3 hours of BAL hemoperfusion was performed day 0 and 3 weeks later on day 21. Biochemical, humoral, and cellular measures of immune response were collected until day 44. The initiation of BAL hemoperfusion was associated with a rapid decline in CH(50) levels of complement and transient neutropenia and thrombocytopenia during all BAL exposures. Xenoreactive antibody response to BAL was increased by use of membranes with large pores and secondary exposures. Skin testing on day 42 showed a delayed-type hypersensitivity response to porcine hepatocytes that also correlated with level of previous antigen exposure. BAL treatment was associated with both immediate and elicited immunologic responses. The immediate response was transient and not influenced by membrane pore size, whereas elicited responses were influenced by pore size of the BAL during previous exposures.