PubMed Health⌕ Search

Biomedical subjects

M A Zieger

Publications and source records attributed to M A Zieger.

At least 19 recordsLinked to original sources
PubMed2001

Improved islet survival and in vitro function using solubilized small intestinal submucosa.

In vitro proliferation of isolated pancreatic islets has become an area of great interest given the scarcity of clinical islet donors and the islet mass requirements for clinical islet transplantation. Small intestinal submucosa (SIS), a naturally occurring extracellular matrix, has been investigated to promote wound healing, tissue remodeling and cell growth. This study evaluated recovery and function of isolated canine pancreatic islets following in vitro tissue culture. Pancreatic islets were isolated from mongrel dogs using standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and EuroFicoll purification. Groups of purified islets were cultured in a humidified atmosphere of 95% air and 5% CO(2) for 48 hours in standard islet culture conditions of CMRL 1066 tissue culture media (Gibco) which had been supplemented with 25microM HEPES, penicillin/streptomycin and either 10% heat inactivated fetal calf serum (FCS, Gibco) or solubilized SIS solution (Cook Biotech, Inc., West Lafayette, IN). The mean recovery of islets following the culture period was determined by sizing duplicate counts of a known volume and viability was assessed by static incubation with low glucose (2.8 mM), high glucose (20 mM) and high glucose solution supplemented with 50 microm IBMX solution. Remaining islets were embedded histologically. From a consecutive series of six culture experiments, a significantly higher (p < 0.05) recovery of islets co-cultured with SIS was observed when compared to controls. Mean islet recovery was 84.5 +/- 2.9% (mean +/- SEM) from the SIS cultured group compared with 64.7 +/- 4.5% from the control group cultured in FCS (p < 0.05, n=6). Islets from the SIS treated group exhibited a significantly higher (p <, 0.05) insulin response to the high glucose stimulus than islets cultured in the standard FCS cultured solution. The calculated stimulation index was 12.3 +/- 3.4 for the SIS-treated group compared with 5.6 +/- 1.8 for the standard cultured group (p < 0.05). The overall mean numbers of islets recovered following in vitro culture was also higher in the SIS-treated group. The proportion of islets with a mean diameter >150 microm increased from 24% to 31% in the SIS-treated group, whereas the same proportion decreased to 18% from 22% in the control (FCS-treated) group. Histological evaluation of fixed tissue samples collected following the culture period identified insulin and glucagon-secreting cells in the SIS and FCS treated groups, however a higher frequency of insulin positive cells were detected consistently in the SIS treated group. A proliferation marker (PCNA) identified positive cells within both groups as well. This study suggests that co-culture of freshly isolated canine islets in medium supplemented with solubilized SIS can improve the post-culture recovery and in vitro islet function. Future investigations will focus on the cellular interactions of SIS, both in vitro and in vivo.

Journal Article↗
PubMed1999

Equations for obtaining melting points for the ternary system ethylene glycol/sodium chloride/water and their application to cryopreservation.

The present study describes the H(2)O-NaCl-ethylene glycol ternary system by using a differential scanning calorimeter to measure melting points (T(m)) of four different ratios (R) of ethylene glycol to NaCl and then devising equations to fit the experimental measurements. Ultimately an equation is derived which characterizes the liquidus surface above the eutectic for any R value in the system. This study focuses on ethylene glycol in part because of recent evidence indicating it may be less toxic to pancreatic islets than Me(2)SO, which is currently used routinely for islet cryopreservation. The resulting physical data and previously determined information regarding the osmotic characteristics of canine pancreatic islets are combined in a mathematical model to describe the volumetric response to equilibrium-rate freezing in varying initial concentrations of ethylene glycol.

Animals↗
PubMed1997

The determination of membrane permeability coefficients of canine pancreatic islet cells and their application to islet cryopreservation.

Sufficient numbers of pancreatic islets for successful allotransplantation can be achieved by storing and then pooling islets from several donors. Optimal MHC matching and infectious disease screening also require long-term storage of islets, and cryopreservation is currently the only practical approach. Cryopreservation protocols may be optimized by modeling the changes in cell volume and the associated damage incurred during cryoprotectant addition and dilution and during cooling and warming. The objective of the present work was to determine the following biophysical parameters of canine islet cells; the osmotically inactive cell volume (Vb), hydraulic conductivity (Lp), cryoprotectant permeability coefficient (Ps), and the reflection coefficient sigma. A determination of these parameters allows the simulation of cell responses using computer models. Islets were isolated by collagenase digestion and Euro-Ficoll purification. After 24 h culture, islets were dissociated into single cells using trypsin and 2 mM EGTA. The kinetic change in cell volume as a function of time after exposure to 2 M dimethyl sulfoxide (Me2SO) was measured using an electronic particle counter at 22, 5, and -3 degrees C. At -11 degrees C, cells were preloaded with 1 M Me2SO and exposed to 4 M Me2SO to prevent the formation of ice in the working solution. Kedem-Katchalsky theory was used to describe the cell volume change kinetics, and a three-parameter curve fitting was performed using the Marquardt-Levenberg method to determine Lp, Ps, and sigma values. The Lp was determined to be 0.19 +/- 0.05, 0.037 +/- 0.005, 0.020 +/- 0.003, and 0.013 +/- 0.005 micron.min-1.atm-1 (mean +/- SD) at 22, 5, -3, and -11 degrees C, respectively. The Ps values were 1.05 +/- 0.50, 0.15 +/- 0.04, 0.096 +/- 0.028, and 0.067 +/- 0.029 x 10(-3) cm.min-1 at 22, 5, -3, and -11 degrees C, respectively. The sigma values were 0.81 +/- 0.16, 0.91 +/- 0.09, 0.80 +/- 0.21, and 0.98 +/- 0.04 at 22, 5, -3, and -11 degrees C, respectively. The temperature dependence or activation energy of Lp and Ps was calculated, using the Arrhenius equation, to be 12.7 and 13.5 kcal.mol-1, respectively. These permeability parameters were used to calculate cell water loss and the likelihood of lethal intracellular freezing during cooling, as well as both water flux and solute concentration gradients across the cell membrane during warming.

Animals↗
PubMed1997

Injury and protection in split-thickness skin after very rapid cooling and warming.

The ability of low glycerol concentrations and high cooling and warming rates to optimize the survival of frozen/thawed split-thickness porcine skin was investigated. 1H nuclear magnetic resonance spectroscopy was used to measure the diffusion kinetics of glycerol in skin at 4, 12, and 22 degrees C. Equilibrium concentrations were 44 to 69% of the external bathing medium. Rate constants for glycerol diffusion (D/l2) were calculated from the uptake data using a plane sheet model and a least squares method and were independent of external glycerol concentrations: D/l2 = 3.84 x 10(-4) 8-1 at 4 degrees C with an activation energy of 11.2 +/- 4.3 kcal/mol. Skin was cooled rapidly (-5100 degrees C/min) after different times of glycerol permeation at 4 or 22 degrees C, and survival was assessed after warming (+5400 degrees C/min) by an oxygen consumption assay. Recovery of aerobic activity increased in a concentration-dependent manner, and reached 100% after a 10-min exposure to 2 M glycerol at 4 degrees C or 3 min at 22 degrees C, for an uptake of 1.1 M glycerol. Light micrographs of freeze-substituted skin showed a glycerol-dependent decrease in the nucleation and growth of ice in the dermis and epidermis after rapid cooling. A 5-mm exposure to 2 M glycerol at 22 degrees C resulted in the elimination of all observable epidermal ice, except for extremely small ice crystals (< or = 0.5 micron diameter) in the intercellular spaces and in few nuclei, and complete preservation of the fibrous structure of dermal collagen bundles. This cryoprotective mechanism has the potential to offer complete protection of both dermal and viable epidermal targets of freeze/thaw injury and may be applicable to other thin, membranous tissues.

Animals↗
PubMed1997

Osmotic characteristics of isolated human and canine pancreatic islets.

Cryopreservation protocols for pancreatic islets may be optimized by modeling the changes in volume that occur during cryoprotectant addition/dilution and cooling/warming. Modeling may be facilitated if it can be demonstrated that isolated islets behave as ideal osmometers where the equilibrium volume is a linear function of the reciprocal of the osmolality of the extracellular medium (the Boyle van't Hoff relationship). The present study was performed to test the hypotheses that: (i) human and canine islets exhibit a linear osmotic response during exposure to hypo-, iso-, and hyperosmotic solutions and to determine the human and canine islets osmotically inactive fraction (Vbp); (ii) to determine if the ionic properties of a concentrated electrolyte solution have an effect on the osmotic response; and (iii) to determine if the osmotic response of pancreatic islets varies between species (human and canine). Islets were isolated from four human and four canine pancreases using standard collagenase digestion and EuroFicoll purification. After 12 h incubation at 37 degrees C, islets (n = 4 from each isolation) were exposed to varying salt or sucrose concentrations (75-1200 mOsm/kg). Islets were held by a micropipette system attached to an inverted microscope, and solutions were exchanged while videotaping. An imaging system was used to determine the cross-sectional area of the islet which was then used to estimate the spherical volume. The results of linear regression analysis indicated that both human and canine islets respond ideally to osmotic conditions within a range of 150-1200 mOsm/kg in both the electrolyte (salt) and the nonelectrolyte (sucrose) solutions. The Vbp value (mean +/- SD) of the four donors was 0.513 +/- 0.068 for canine islets in sucrose solutions, 0.474 +/- 0.068 for canine islets in salt solutions, 0. 425 +/- 0.073 for human islets in sucrose solutions, and 0.39 +/- 0. 073 for human islets in salt solutions. There was a significant difference between human and canine islets (P < 0.05) but not between experimental solutes (NaCl or sucrose) (P < 0.05). These data may be used in conjunction with osmotic tolerance data to develop improved methods for the cryopreservation of isolated islets.

Animals↗
PubMed1996

Mechanisms of cryoinjury and cryoprotection in split-thickness skin.

Successful cryopreservation of tissues will ultimately require a more detailed understanding of how the in situ environment modifies cell responses during cooling and warming. Low-temperature responses of porcine split-thickness skin and isolated basal keratinocytes were compared after various cooling protocols and in the presence and absence of cryoprotectants. Recovery was assessed by measuring oxygen consumption kinetics in skin and tetrazolium reduction in isolated cells. Freeze substitution was used to visualize ice nucleation and growth in skin. The results indicated that the time required for diffusion of water in split-thickness skin delayed osmotic responses in the basal keratinocytes and resulted in increased intracellular and intercellular ice formation. Rapid cooling (-200 degrees C/min) in the presence of cryoprotectants resulted in a reduction in the number of cells containing ice and the size of the intercellular ice crystals and an increase in tissue recovery. These observations support other reports which suggest that cell-to-cell and cell-to-substrate interactions are sensitive sites for cryoinjury. A practical recommendation from this study is that high recovery of split-thickness skin may be achieved with protocols using high cooling rates.

Animals↗
PubMed1991

Factors influencing survival of mammalian cells exposed to hypothermia. V. Effects of hepes, free radicals, and H2O2 under light and dark conditions.

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light ("dark conditions") or by supplementing the medium with 10 micrograms/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under "lighted conditions." No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 degrees C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 micrograms/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 micrograms/ml catalase does not protect cells from hypothermic (5 and 10 degrees C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.

Animals↗
PubMed1990

Factors influencing survival of mammalian cells exposed to hypothermia. IV. Effects of iron chelation.

Survival of V-79 Chinese hamster cells was assessed by colony growth assay after hypothermic exposure in the presence of iron chelators. At 5 degrees C, maximum protection from hypothermic damage was achieved with a 50 microM concentration of the intracellular ferric iron chelator Desferal. A 3-hr prehypothermic incubation with 50 microM Desferal followed by replacement with chelator-free medium at 5 degrees C also provided some protection. This was not observed when the extracellular chelator DETA-PAC (50 microM) was used prior to cold storage. Treating 5 degrees C-stored cells with Desferal just prior to rewarming was ineffective, but treating cells with Desferal during hypothermia exposure after a significant period of unprotected cold exposure ultimately increased the surviving fraction. Submaximal protection during hypothermia was achieved to various degrees with extracellular chelators at 5 degrees C, including 50 microM DETAPAC and 110 microM EDTA. EGTA (110 microM) had little effect. The sensitization of cells at 5 degrees C with 200 microM FeCl3 could be reduced or eliminated with Desferal in accordance with a 1:1 binding ratio. At 10 degrees C, 50 microM Desferal, 50 microM DETAPAC, and 110 microM EDTA were as or less effective in protecting cells than at 5 degrees C. An Arrhenius plot of cell inactivation rates shows a break at 7-8 degrees C, corresponding to maximum survival for control cells and cells in 50 microM Desferal; however, the amount of protection offered by the chelator increases with decreasing temperature below about 19 degrees C, and sensitization increases above that point. It has not previously been shown that iron chelators protect against cellular hypothermia damage which is uncomplicated by previous or simultaneous ischemia. This may be relevant to the low-temperature storage of transplant organs, in which iron of intracellular origin and in the perfusate may be active and damaging.

Animals↗
PubMedDate not supplied

A simple, effective system for assessing viability in split-thickness skin with the use of oxygen consumption.

To address the recognized need for better viability-assessment techniques in the banking of allograft skin, we have developed a simple, effective, and easily constructed system for measuring the aerobic activity in small skin samples. The system consists of a microcomputer interfaced to a Clark-type polarographic oxygen electrode to measure oxygen concentrations in discs (5 mm diameter) of split-thickness skin. The system allows simultaneous data acquisition and an on-screen display of oxygen readings on a linear or logarithmic scale. The computer program quantifies the rate of oxygen consumption by calculating a linear regression of oxygen measurements between 100 and 600 seconds. Data are stored in a standard format for easy transfer to commercial software packages for further analysis. The kinetics of oxygen consumption by porcine split-thickness skin stored at 4 degrees C for up to 21 days was assessed with this technique and showed a gradual decline in rate as function of storage time, reaching 50% recovery after 6 days. This assay is a simple and inexpensive method to establish and perform, thus making it suitable as a routine assay for use in the banking of skin and other tissues.

Animals↗
PubMedDate not supplied

Hypoosmotic exposure of canine pancreatic digest as a means to purify islet tissue.

The development of more effective means to separate pancreatic islets from the unwanted exocrine tissue would greatly advance the field of clinical islet allotransplantation in the treatment of insulin-dependent diabetes mellitus. Recent experiments with hamster islets have demonstrated a selective destruction of dissociated single exocrine cells when exposed to hypotonic conditions. It was the aim of this study to extend these observations to the canine model with collagenase dissociated pancreatic tissue and to evaluate the treatment's effect on islet function. Pancreases from five mongrel dogs were digested using an automated protocol of intraductal delivery of collagenase, and gentle dissociation. Duplicate samples of pancreatic digest were removed for insulin and amylase determination prior to and immediately following exposure to 50 mOsm/kg salt solution for a period of 30, 60, or 300 s before returning the digest to isoosmotic conditions. The remaining digest was cultured for a period of 48 h at 37 degrees C before the tissue was recombined, washed, and a third sample removed for insulin and amylase. In vitro viability was then assessed using a static incubation assay with insulin content measured using a double-antibody radioimmunoassay, and amylase was determined using a colorimetric assay system. No difference in the insulin or amylase levels between the experimental groups was observed immediately following the hypotonic exposure; however, a significant decrease in the amylase content was observed following the 48-h culture period in digest that had been hypoosmotically exposed for 60 or 300 s compared with the pretreatment group (2.83 +/- 0.41 IU amylase/mg pancreas vs. 1.29 +/- 0.21 and 0.83 +/- 0.12, mean +/- SEM, p < 0.05). Insulin content was also significantly reduced in the 300-s exposure group compared with nontreated controls (3.2 +/- 0.6 mU insulin/mg pancreas vs. 2.0 +/- 0.2). The insulin/ amylase ratio (I/A), a measure of islet and exocrine content, was 1.1 +/- 0.13 following pancreas dissociation and 1.34 +/- 0.21 for control tissue cultured for 48 h. The I/A ratio increased following hypoosmotic exposure to 1.50 +/- 0.31 for tissue exposed for 30 s, 1.77 +/- 0.19 for 60-s exposure, and 2.54 +/- 0.13 for tissue exposed for 300 s (p < 0.05, vs. pretreatment group). In vitro insulin secretion was equivalent with the exception of the tissue exposed for 300 s, which had an increased basal level of insulin resulting in a significantly decreased stimulation index (3.8 +/- 0.5 vs. 8.1 +/- 1.2 for the purified islet control group, p < 0.05). These results suggest that a brief hypotonic exposure to pancreatic digest can alter the insulin/amylase ratio; however, there is a functional impairment on subsequent islet function after a period of in vitro tissue culture.

Animals↗