Frozen-thawed Suspensions Research Articles

Effect of pH, ionic strength, and freezing treatment on a colloidal suspension of egg white aggregates

• pH and ionic strength effected egg white suspensions under frozen condition. • Number of egg white aggregates decreased during freezing process. • Surface characteristic was modified by freezing, pH, and ionic strength. • Modification of egg white aggregation affected foam stability. Characteristics of protein colloidal suspensions can be modified by freezing treatment. In this study, different colloidal suspensions of egg white aggregates (with different pH levels and ionic strengths) were frozen for several days, after which the colloidal suspensions were analyzed by a tunable resistive pulse sensing device to detect changes in particle characteristics. The average particle diameter in suspension increased with an enhancement in ionic strength. Particle number concentration decreased during five days of freezing treatment. The same colloidal suspensions were also analyzed using a small-angle X-ray scattering device. The results indicate that the inner structure was influenced by pH but did not change during the freezing process. The stability of foam made from frozen-thawed suspensions also increased with the enhancement of ionic strength. Based on these results, it appears that by controlling pH levels, ionic strength, and freezing time, the characteristics of egg white aggregates can be modified by freezing.

The efficiency of magnetic-activated cell sorting and fluorescence-activated cell sorting in the decontamination of testicular cell suspensions in cancer patients

Before clinical application, the feasibility and safety of autologous testicular stem cell transplantation should be explored. Apart from limitations in their numbers, spermatogonial stem cells may also be contaminated by malignant cells. Therefore, both enrichment and decontamination before transplantation may be necessary. This study aimed at evaluating the decontaminating potential of magnetic-activated cell sorting (MACS) and/or fluorescence-activated cell sorting (FACS) for both murine and human testicular cell suspensions. In the mouse, the effectiveness of the transplantation technique after cell sorting was also assessed. Murine testicular cells were contaminated with 5% EL4 cells. Fresh and frozen-thawed suspensions were sorted using MACS (CD49f (+)) and FACS (CD49f(+), H-2Kb(-)) and evaluated by FACS, cell culture and transplantation into W/W(v) mice. Human testicular cells were contaminated with 5 or 0.05% CCRF-SB (SB) cells. Frozen-thawed suspensions were sorted using FACS (HLA class I(-)) and evaluated by FACS, cell culture and PCR for the B-cell receptor. In the mouse, the sorted fractions contained 0.39% H-2K(b)-positive and 76.55% CD49f-positive cells. After transplantation, 1 in 20 recipient mice developed a malignancy. In the human experiments, an average of 0.58% SB cells was detected after sorting. In only 1 of 11 samples, there were no SB cells observed. MACS and/or FACS are insufficient for completely depleting testicular tissue of malignant cells. Although more research on alternative decontamination techniques is necessary, developing a reliable method to screen a priori testicular tissue for malignant cells may be equally important.

Recovery, survival and functional evaluation by transplantation of frozen-thawed mouse germ cells.

Establishing a successful method for testicular stem cell transplantation of frozen-thawed testicular cells would be of immense benefit to boys with childhood cancer undergoing a sterilizing treatment. In this study, we evaluated different cryopreservation protocols in a mouse model by means of testicular germ cell transplantation (TGCT), in order to establish an optimal freezing protocol. In a first series of experiments, we compared an uncontrolled protocol with 1.5 mol/l dimethyl sulphoxide (DMSO) versus a controlled long protocol (cooling to -80 degrees C) and observed a better viability with the latter protocol (36% versus 48%, P < 0.05). We then compared survival after two thawing methods (37 degrees C water versus ice water) in either a DMSO- or an ethylene glycol (EG)-based protocol, and found no difference. In order to evaluate the functional capacity of the cryopreserved testicular suspension, TGCT was performed with both fresh and frozen-thawed suspensions. In 90% of the successfully injected testes, spermatogenesis was reinitiated using fresh suspensions. In contrast, this figure was only 12.5 and 22.7% after cryopreservation, for the short controlled EG protocol and the uncontrolled DMSO protocol, respectively. Reinitiation of spermatogenesis is possible after cryopreservation of testicular germ cell suspensions. Although cell survival was acceptable, our results after TGCT show that our protocols need further improvement.

Cryoprotection of murine lymphocyte subpopulations using a microprocessor-controlled cooling system

Single-cell suspensions of splenic lymphocytes from 5- to 6-month-old C57BL/6 mice were cryopreserved using cooling rates ranging from −0.25 to −10.0 °C/min with the microprocessor-controlled cooling system developed in our laboratory. The cells (30 × 10 6 cells/ml) were suspended in RPMI 1640 containing 10% FCS and 10% DMSO, and a total volume of 1.75 ml was frozen. Fluorescein-diacetate staining identified viable cells in unfrozen controls and frozen-thawed suspensions. Functional capacity was assessed in vitro by the incorporation of [ 3H]thymidine by dividing cells activated with graded concentrations of the T-lymphocyte mitogens, PHA-P and Con A, and the B-lymphocyte mitogen, LPS. High percentages of viable cells were recovered after cooling at rates ranging from −0.5 to −5.0 °C/min, as compared with those of unfrozen control suspensions. Incorporation of [ 3H]thymidine by T and B cells reached similar levels after cooling at rates ranging from −0.25 to −5.0 °C/min, and the percentage incorporation of [ 3H]thymidine as compared with that of unfrozen cells was 80–100%, except for T lymphocytes activated with PHA-P after cooling at −5.0 °C/min. The relative response of cell suspensions to T- and B-cell mitogens, the relative mitogenic index, was unchanged from that of unfrozen controls in suspensions cooled at all rates including two (−0.25 and −10.0 °C/min), which permitted recovery of only 55% of unfrozen cells. The importance of the constant cooling rates and rapid compensation for heat released at the phase change using the microprocessor-controlled system and of precise determinations of cellular viability and function are discussed and related to the apparent protection conferred on subpopulations of murine lymphocytes using cooling rates ranging from − 0.25 to −10.0 °C/min.

Cause of Microbial Death during Freezing in a Soft-Serve Ice Cream Freezer

Death of microbial suspensions during freezing with agitation was studied by following the optical density changes of yeast suspensions in frozen-thawed sucrose solutions; and by cytological examination of frozen-thawed yeast suspensions using a light microscope, and frozen-thawed suspensions of Escherichia coli with an electron microscope.Changes in the optical density values of frozen-thawed yeast suspensions with duration of freezing, and in the supernatant liquid of these suspensions after centrifugation, suggested changes in the size distribution of the suspended cells and the presence of cytoplasmic materials in the supernatant layer.Photomicrographs established that disintegration of yeast cells occurred during freezing with agitation. Electron micrographs revealed the disintegration of cells of E. coli.