Retaining ionic concentrations during in vitro storage of tissue for microanalytical studies

Area of Science:

• Biochemistry
• Cell Biology
• Analytical Chemistry

Background:

• Investigating human or animal tissue using X-ray microanalysis requires tissue preservation between removal and freezing.
• Standard buffer solutions can alter cellular elemental concentrations, compromising analysis accuracy.
• A need exists for storage buffers that maintain in situ elemental concentrations.

Purpose of the Study:

• To develop and optimize storage buffer solutions that minimize changes in cellular elemental concentrations in excised tissues.
• To evaluate the efficacy of modified Krebs-Ringer solutions in preserving elemental homeostasis in rat liver and submandibular gland tissues.

Main Methods:

• Preparation of modified Krebs-Ringer solutions by adjusting NaCl, KCl, and K-gluconate concentrations.
• Incubation of excised rat liver and submandibular gland tissues in various modified solutions at 4°C for up to 6 hours.
• Analysis of cellular elemental concentrations (Na, K, Ca, S, Mg, P, Cl) and cell volume changes.
• Assessment of cell membrane integrity using trypan blue uptake and functional response to cholinergic stimulation.

Main Results:

• Specific modified Krebs-Ringer solutions (e.g., 85% high K+ for liver, 80% high K+ for submandibular gland) demonstrated optimal retention of intracellular elements (Na, K, Ca, S, Mg).
• Modified buffers maintained cellular elemental concentrations closer to in situ values compared to standard high K+ solutions.
• Reduced cell swelling (edema) and improved cell membrane integrity (lower trypan blue uptake) were observed in tissues stored in modified solutions.
• Preserved submandibular gland tissue in modified solutions retained the ability to exhibit chloride efflux upon cholinergic stimulation.

Conclusions:

• Optimized Krebs-Ringer solutions with specific K+, Na+, and Cl- concentrations can effectively preserve elemental homeostasis in excised rat tissues.
• These modified buffers offer significant advantages over standard solutions by minimizing elemental shifts, cell volume changes, and maintaining membrane integrity.
• The developed storage solutions are suitable for preparing tissues for X-ray microanalysis, ensuring more reliable and accurate results.