Days Of Postmortem Storage Research Articles

PSVI-7 Postmortem in Vitro Culture of Cells from Visceral Organs Stored at 4°C in Small Ruminants

Abstract Exploration of cell viability and their regenerative capacity in postmortem tissues has a huge potential for tissue/organ transplantation and cell therapies in both human and animals. Proliferative stem cells have been recovered from refrigerated postmortem skin tissue in bovine, goat and sheep, and from ligaments in horses. However, it is not clearly known whether live and proliferative cells can be recovered from visceral organs that stay comparatively warmer than other tissues after death, and whether the recovery time can be extended after refrigeration of individual organs. To achieve this goal, six different organ tissues i.e. lung, heart, oviduct, liver, kidney and skin from goats were procured from slaughter house and stored in the laboratory at 4°C. Ten small sized (2-3mm2) explants from each of the five organs were then cultured in two 35mm dishes for each time point in DMEM media supplemented with 10% fetal bovine serum. The tissues were cultured on day 0, 3, 6, 9 and 12. The dishes were observed under inverted microscope and any explant exhibiting outgrowth of cells on day 10-12 of culture was considered positive. Our results exhibited outgrowth of cells around 3 (lung, oviduct, skin) of the 6 organs tested. Kidney, liver and heart tissues did not show any outgrowth. Interestingly, oviduct and skin, tissues exhibited outgrowth even after 9 days of postmortem storage in refrigerator, whereas lung tissues show growth only up to 6 days of storage. Experiments to test the cell survival limits on more animals of different age groups and characterization of recovered cells are underway.

PSX-B-16 Recovery of live cells from refrigerated sheep skin after different days of cold storage

Abstract Cryopreservation of tissues from domesticated and wild relatives has been suggested to conserve genetic diversity. Ensuring that the tissues have live cells prior to preservation, especially in postmortem tissues, is an essential stem for success. How long cells live after clinical death is not precisely known in animals. The objective of this study was to evaluate the limits of cell survival in sheep skin stored at 4°C postmortem. Ear skin was procured from six random but healthy slaughtered animals and stored at 4°C in the lab. Ten explants (2–3 mm2) were cultured from each animal in DMEM media with 10% FBS, 50 units/mL of penicillin, 50 µg/mL of streptomycin, and 2.5 µg/mL of fungizone on two 60 mm dishes after 0, 10, 20, 27, 30, 35, 38, 41, 45, 50, 55, 60, 65 and 70 days of storage. Outgrowth of fibroblast-like cells around the explants was scored after 10 days of culture in a CO2 incubator. Results show outgrowth of cells up to 65 days of postmortem storage. Out of 476 explants adhered to dish surface, 374 (78.58%) exhibited outgrowth. The number of outgrowing cells decreased with increasing postmortem storage time interval. To test the differences between cell cultures obtained from postmortem fresh and stored tissues, we established secondary cultures from primary cells of 0-dpm and 65-dpm time points from selected cell lines. Both cultures exhibited similar growth morphology and growth curve, could be cryopreserved with >80% post freezing cell viability, lasted in cultures up to 35 passages, and expressed GFP gene upon transfection with a GFP gene containing plasmid vector. The karyotype analysis of 65-dpm tissue derived cells revealed a normal female karyotype without any genetic aberrations. These results suggest that normal proliferative cells can be recovered from sheep skin up to about 2 months postmortem, if kept refrigerated.

Sarcoplasmic and myofibril-bound calpains during storage of pork longissimus muscle: New insights on protein degradation

The subcellular distribution of calpain-1 and -2 and the proteolytical activity of myofibril-bound calpains in pork were investigated during 12 days cold storage. The content of sarcoplasmic calpain-1 decreased during storage while myofibril-bound calpain-1 content first increased (P < 0.05) to 17% of that of 12 h-sarcoplasmic calpain-1 on day 6 followed by a gradual decrease with subsequent storage, suggesting that calpain-1 gradually translocated from sarcoplasm to myofibrils during the initial 6 days of postmortem storage. Intact desmin decreased (P < 0.05) after incubation of myofibrils with 0.05 mM Ca2+, and this was more pronounced with 5 mM Ca2+ (P < 0.05). Ca2+ titration curves of day 6 myofibrils showed two distinct proteolytic activities becoming activated in the range 0.03 to 0.06 mM and 0.4 to 0.8 mM Ca2+, respectively. The results suggest that both calpain-1 and calpain-2 binds to myofibrils during storage and subsequently degrade structural proteins including desmin.

Recovery of Fibroblast-Like Cells after 160 Days of Postmortem Storage of Goat Skin Tissues in Refrigerated Media

Animals have been cloned from frozen decade old postmortem tissues preserved within few hours of animal death. Delay in tissue preservation may reduce cloning success due to compromised nuclear DNA integrity. In vitro culture of cells ensures nuclear integrity and is a preferred method of preparing somatic cells for cloning. However, the time limits of postmortem recovery of cells capable of in vitro culture are not precisely known. Here we show recovery of fibroblast-like cells after 160 days of postmortem storage of goat skin in culture media at 4?C. Forty skin explants were cultured at 10 days interval up to 160 days and the outgrowing fibroblast-like cells around them were observed under inverted microscope. Explants with a cluster of more than 50 cells, after 10-12 days of culture initiation, were considered positive. We observed the outgrowth in all the time points, however, the confluence level reduced with increasing postmortem time interval. Secondary cultures established from primary outgrowth of 0-, 90- and 160-dpm tissues exhibited similar fibroblast-like morphology. Growth curves of 0- and 90-dpm cells were similar but 160-dpm cells grow slightly slower.Cytogenetic analysis performed on twenty G-banded metaphase cells of 160-dpm cell line revealed an apparently normal male goat karyotype with 60 chromosomes and their post-freezing cell-viability was >67%. Potential of using these cells to clone the goats remains to be seen in future. These findings may be useful in decisions for preservation of tissues for future cloning of animals and cell therapy.

Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle

Calpains are Ca2+-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature

Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85% explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67%) and 6 days (13.3%) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.

Changes in proteasome activity during postmortem aging of bovine muscle

Changes in the chymotrypsin-like, trypsin-like, peptidylglutamylpeptide hydrolyzing and caseinolytic activities of proteasomes in bovine rectus abdominis muscle were measured during the first seven days of postmortem storage. Enzyme assays were performed in crude extracts under near-physiological conditions, since the activities are likely to be altered by purification. The different proteasome activities at cellular pH were stable at different times postmortem, and were 40, 76, 50 and 61% of their at-death value after 7 days of storage at 4 °C. This considerable postmortem stability of proteasome activities, despite the marked decrease in pH, allows them to play a role in meat tenderization in synergy with other proteolytic systems.

Nitric oxide synthase activity in muscle foods

Nitric oxide is enzymatically produced in animals by nitric oxide synthase (NOS). Nitric oxide reacts with superoxide to form peroxynitrite, which initiates oxidative reactions. To assess the potential for nitric oxide formation in muscle, NOS activity was determined in fresh muscle (<8 h post-mortem) from several species under conditions expected in muscle foods. Fresh muscle from all species exhibited NOS activity. NOS activity in muscle was reduced during refrigerated storage. Chicken thigh muscle NOS activity was not affected by pH over the range of 4.5–7.4, but was inhibited by 1 and 2% NaCl. Chicken thigh muscle NOS activity was stimulated at internal cooking temperatures up to 55 °C but was completely inhibited at higher temperatures. Results of this study indicate that post-mortem NOS is only active for the first several days of post-mortem storage.

Combination of low voltage electrical stimulation and early postmortem temperature conditioning on degradation of myofibrillar proteins in Korean native cattle (Hanwoo)

The combination effect of low voltage electrical stimulation (LVES) and early postmortem (PM) temperature conditioning (2, 16, and 30°C until 3 h PM) on degradation of myofibrillar proteins were determined from Korean native cattle (Hanwoo). Myofibrils were removed at 1, 2, 3, 7, and 14 days of PM storage (2°C) and analyzed for titin, nebulin, desmin, and troponin-T by SDS-PAGE and by Western blot analysis. Degradation rate of myofibrillar proteins was affected by the combination of LVES and temperature conditioning. LVES-30°C treatment resulted in faster degradation of titin, nebulin, desmin, and troponin-T during PM storage than the other treatments. Degradation of titin took place more slowly than nebulin, desmin or troponin-T.

Desmin Degradation in Postmortem Fish Muscle

ABSTRACTThe degradation of desmin was studied in postmortem white dorsal muscle from sea bass, brown trout, turbot and sardine, stored at 0–4°C. Based on desmin specific antibody tests, results showed that the extent and rate of desmin degradation in postmortem fish muscle varied considerably among species. There was no degradation of desmin during the first 4 days postmortem storage of sea bass and brown trout, whereas 10 to 20% of muscle desmin was degraded during the first 4 days of postmortem storage of turbot. Sardine muscle presented a very complex pattern of desmin degradation products and aggregated desmin fragments detected within the first 24h. Most polypeptides were larger than the nondenatured desmin. Desmin degradation would not be a suitable marker for evaluation of postmortem changes of stored fish.

Postmortem Degradation of Titin and Nebulin of Beef Steaks Varying in Tenderness

ABSTRACTPurified myofibrils were isolated from “tender” and “less‐tender” bovine longissimus muscle at death and at 1, 3, 7, and 14 days of postmortem storage (4oC). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) was used to detect changes in the myofibrillar/cytoskeletal proteins, titin and nebulin. Titin and nebulin bands were observed to be less intense on gels from “tender” than from “less‐tender” steaks. These results suggest that titin and nebulin were more rapidly degraded in “tender” than in “less‐tender” steaks, and that the extent of beef loin steak tenderness may be dependent upon the postmortem degradation of titin and nebulin.

Effect of Postmortem Storage on Cold-Shortened Bovine Muscle: Analysis by SDS-Polyacrylamide Gel Electrophoresis

Myofibrils were isolated from the longissimus (L) muscle of control (CON) and cold-shortened (CS) muscles after 0, 1, 3, 7, and 10 days of postmortem storage at 2°C. Isolated myofibrils were then examined by SDS-polyacrylamide gel electrophoresis to monitor the changes in the myofibrillar proteins during postmortem storage. The main changes in CS muscles were the gradual appearance of 110,000-, 95,000-, and 30,000d-dalton components and the disappearance of desmin and troponin-T components of myofibrils. In addition, there was a gradual increase in the intensity of a protein around 55,000-daltons. CON samples showed similar changes to those of CS samples. It appears that myofibrillar proteins of cold-shortened muscles are affected by postmortem aging in a manner similar to that of the normally chilled muscles.

Effects of Postmortem Storage on Muscle Protein Degradation: Analysis by SDS-Polyacrylamide Gel Electrophoresis

Changes in myofibrillar proteins of bovine longissimus and semitendinosus muscles were examined during 14 days of postmortem storage at 2°C by SDS-Polyacrylamide gel electrophoresis. Major changes in both muscles were: (1) appearance of a 95,000-dalton component; (2) gradual disappearance of troponin-T and gradual appearance of a 30,000-dalton component; (3) gradual increase in intensity of a protein around 55,000-daltons; and (4) gradual appearance of a 110,000-dalton component. With the exception of the 95,000-dalton component, the other changes have been reported previously by other authors. At the present time we have no knowledge of the origin of the 95,000-dalton component, but it is known that calcium-activated factor (CAF) is responsible for degradation of troponin-T. Appearance of the 95,000-dalton component during postmortem storage suggests involvement of CAF for increasing tenderness during postmortem storage.

Effect of Postmortem Storage on Degradation of the Myofibrillar Protein Titin in Bovine Longissimus Muscle

ABSTRACTPurified bovine longissimus muscle myofibrils were prepared from muscle at death and from muscle samples stored at 2°, 25°, or 37°C for 1, 3, and 7 days postmortem. Tbe myofibrils were analyzed by sodium dodecyl sulfate‐polyacrylamide slab gel electrophoresis. Titin migrated as a closely spaced doublet of very high molecular weight (Mr∼ 1 × 106) in myofibrils from at‐death muscle samples. With increased storage time and temperature, the top band of the titin doublet gradually disappeared. the lower doublet band (putative breakdown product of upper band) remained after 7 days storage at 2° or 25°C, but disappeared by 3 days of postmortem storage at 37°C. Thus, titin is degraded in postmortem muscle, and the rate of degradation is enhanced by increases in storage time and temperature.

Human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts in primary cell culture. A comparative study of growth characteristics, morphology, and phagocytic activity by light and scanning electron microscopy

To obtain baseline data for our experimental investigations in vitro we established a pure cellline culture model of human trabecular endothelium, corneal endothelium, keratocytes, and scleral fibroblasts, using an identical environment for each cell type. We studied the growth characteristics, morphology and phagocytic activity of live cultures by phase-contrast microscopy and time-lapse cinephotomicrography; fixed preparations were examined by light and scanning electron microscopy. From these observations, we were able to characterize the distinctive features of the cells. By correlated ultrastructural and tissue culture studies, we showed that human cadaver eyes cold-stored (4°C) up to 5 days can be used as a tissue source of trabecular endothelium, keratocytes and scleral fibroblasts. Corneal endothelium, however, rarely survived after 4 days of postmortem storage. Our observations revealed that, in primary confluent culture, the trabecular cells grew in a monolayer. Individual cells had flattened, elongated cell bodies, a centrally located, oval nucleus, and a generally smooth cell surface with occasional microvillous projections; in places, adjacent cells and their processes adhered closely one to another. Corneal endothelium at confluency presented a flattened hexagonal profile of cells arranged in a honeycomb pattern, interdigitating cell borders, a centrally located nucleus, prominent perinuclear microvillous projections, and numerous surface invaginations. The keratocytes and scleral fibroblasts had a thin and elongated, spindle-shaped profile with a remarkably smooth cell surface and an oval to oblong nucleus surrounded laterally by scanty cytoplasm; the cell surfaces and processes frequently overlapped but rarely formed firm attachments. In contrast to the monolayer growth of trabecular and corneal endothelium, these cells characteristically grew in superimposed layers. In primary culture and with latex spheres and carmine particles used as markers, the trabecularendothelial cells showed a greater phagocytic capability than did corneal endothelium, keratocytes and scleral fibroblasts. Our preliminary studies indicate that cytochalasin B markedly inhibits the phagocytic activity of the trabecular cells.

EFFECT OF MATURITY AND MARBLING ON THE MYOFIBRIL FRAGMENTATION INDEX OF BOVINE LONGISSIMUS MUSCLE

ABSTRACTMyofibril fragmentation index (MFI), sarcomere length, total and soluble collagen, Warner‐Bratzler shear and sensory tenderness were determined on loin steaks from carcasses that varied in marbling, maturity and composition. Loin steaks from 72 A, 11 B, 19 C and 22 E maturity beef carcasses were removed at lo‐14 days of postmortem storage at 2°C. Steaks were double wrapped in freezer paper and stored at ‐29°C until the samples were used for analysis. MFI was significantly related to sensory tenderness scores as evidenced by the correlation coefficients of 0.66, 0.77, 0.75 and 0.71 for A, B, C and E maturities, respectively, and 0.76 for all maturities. MFI of loin steaks from A maturity were significantly different from MFI of loin steaks from B, C and E maturity carcasses. Differences in MFI among marbling groups within A maturity were found only at the lowest degree of marbling (practically devoid). There fore, it seems that MFI could be useful as a criterion of quality for A, B, C and E maturity carcasses, and especially A maturity carcasses without being significantly affected by marbling degree or carcass composition. These results further substantiate the use of myofibril fragmentation tenderness as a term to describe a state of tenderness of conventionally aged bovine longissimus muscle.