Cryopreservation and hypothermic storage of lacrimal gland: towards enabling delivery of regenerative medicine therapies for treatment of dry eye syndrome.

Severe dry eye syndrome (DES) can cause painful loss of vision and may result from lacrimal gland dysfunction. Current treatments are palliative, so a causative therapy is desirable. The ability to (cryo)preserve lacrimal gland tissue or epithelial cells would simplify this. Here, lacrimal gland tissue was cryopreserved in 10% dimethylsulphoxide in liquid nitrogen, or stored at 4 °C in culture medium for up to 7 days, and compared with fresh tissue using immunohistochemistry. Cultures were initiated from fresh and stored tissue, and cells characterised in P1 for proliferation (WST-1), colony-forming efficiency (CFE) and secretory capacity (immunocytochemistry and ß-hexosaminidase activity assay). Tissue stored for > 3 days at 4 °C displayed grossly altered tissue architecture when compared with fresh tissue, decreased acinus density and increased caspase-3 activity. Cryopreserved tissue showed less obvious signs of damage without caspase-3 activation. Storage at 4 °C and cryopreservation delayed epithelial outgrowth compared with that from fresh tissue initially (p  < â€…0.05) but, by day 9, all explants showed comparable outgrowth (~90%), except tissue stored at 4 °C for 3 or 7 days (p  < â€…0.05 compared with fresh tissue). Epithelial cell yields per explant were similar from fresh and stored tissue, apart from tissue stored at 4 °C for 7 days (p  < â€…0.01). In P1, epithelial cells from fresh and stored tissue were largely equivalent in terms of: proliferation; CFE (~21%); Rab3D, HexA and lysozyme expression; mucin production; and ß-hexosaminidase activity. These data demonstrate that cryo(preservation) of lacrimal gland tissue and cells is possible, which may enable use of autologous cells in regenerative medicine approaches to treating DES. Copyright © 2016 John Wiley & Sons, Ltd.

Cryopreservation of encapsulated liver spheroids using a cryogen-free cooler: high functional recovery using a multi-step cooling profile.

Acute liver failure has high mortality with unpredictable onset. A bioartificial liver, comprising alginate-encapsulated HepG2 spheroids, could temporarily replace liver function but must be cryopreservable. For clinical use, contamination risks from liquid coolants for cryopreservation and storage should be minimized. A cryogen-free cooler was compared to nitrogen vapour-controlled cryopreservation of alginate-encapsulated liver cell spheroids (AELS). AELS were cooled using a multi-step, slow-cooling profile in 12 percent v/v Me2SO Celsior and stored in liquid nitrogen; temperatures were recorded throughout, and the AELS were assayed at 24, 48 and 72 hours post-warming and results compared to unfrozen control values. Viability was assessed by fluorescent staining and quantified using image analysis; cell numbers were quantified using nuclear counts, and cell function using albumin synthesis. The cryogen-free cooler performed the cooling profile as desired, apart from one step requiring a rapid cool ramp. Viability, cell numbers and function were similarly decreased in both cryopreserved groups to about 90 percent, 70 percent and 65 percent of the controls respectively. This technology offers a clinic alternative to liquid nitrogen-coolant cryopreservation.

Private dentistry: why we need the facts.

Rumor and speculation abound within the dental profession about practitioners withdrawing from the NHS to deliver more private dentistry. Due to an absence of effective monitoring or research into this issue the real situation is unclear. We decided to find out what proportions of the gross incomes of general dental practitioners in the East Riding Health Authority were generated by private dentistry. We also sought to establish if they perceived any differences between the quality of their private and NHS work. Our findings and the issues raised are considered for general dental practitioners, for people residing in the authority, and for managers and policy makers. We conclude that the effective management of the supply of NHS dentistry should include a method of systematic monitoring of trends in the delivery of private dental services and the impact on the availability of NHS care. Effective measures are also needed to influence the number and location of dentists in health authorities in England and Wales to ensure adequate and equitable access to NHS dentistry.

Destruction of endogenous low density lipoprotein in incubated intima.

The effect of incubation on the content of endogenous intact plasma lipoprotein (LP) has been examined in minced samples of normal intima and lesions from 38 patients. Both the electrophoretically mobile and the immobilized LP fractions decreased on incubation, and the rate of destruction was proportional to LP concentration (r=0.832, p less than 0.001). Mincing the intima with EDTA before incubation increased the rate of destruction about 4-fold in fibrous lesions but not in lesions containing numerous fat-filled cells. The destruction of LP was highly dependent on pH; the rate was highest below pH 5.5 and destruction was almost completely inhibited above pH 6.4. In standard cathepsin assays haemoglobin substrate was hydrolysed at a rate comparable to the rate of destruction of LP. The results suggest that LP may be degraded by a lysosomal cathepsin in intima.

The release of an immobilized lipoprotein fraction from atherosclerotic lesions by incubation with plasmin.

A large amount of plasma low density lipoprotein is present in human aortic intima, and this can be removed and measured by electrophoresis directly from the minced tissue into an antibody-containing gel. We now find that, in addition to this electrophoretically mobile lipoprotein, there is an immobilized lipoprotein fraction than can be released from lesions by incubation of the tissue sample with plasmin or other proteolytic enzymes after the mobile lipoprotein has been removed. The concentration of immobilized lipoprotein is highly correlated with the concentration of the residual cholesterol (not mobile on electrophoresis) that has accumulated in the tissue (r = 0.702; P less than 0.001). Thus, in normal intima and early gelatinous lesions it is about 15% of the concentration of mobile lipoprotein, whereas in the atheroma lipid layers of fibrous or gelatinous plaques it may be 2 or 3 times greater than the concentration of mobile lipoprotein. This suggests that immobilization of plasma lipoprotein is an intermediate step in the irreversible deposition of extracellular cholesterol in atherosclerotic lesions. Incubation with plasmin allowed maximum release of lipoprotein: plasmin = crude collagenase greater than trypsin greater than "pure" collagenase greater than chondroitinase ABC in order of their relative effectiveness. The concentration of immobilized lipoprotein was significantly correlated (r = 0.793; P less than 0.001) with the concentration in the tissue of fibrin or other insoluble derivatives of fibrinogen ("fibrin"). In aliquots of lesions incubated with varying amounts of plasmin for varying times there was a constant relation between release of lipoprotein and release of fibrin-degradation products. Together, these findings suggest that the lipoprotein is associated with insoluble "fibrin". This appears to be of considerable clinical interest, suggesting a synergism between lipoprotein and fibrinogen in the accumulation of lipid in lesions.

Insoluble "fibrin" in human aortic intima. Quantitative studies on the relationship between insoluble "fibrin", soluble fibrinogen and low density lipoprotein.

A quantitative assay for fibrin or other insoluble fibrin-like antigens ("fibrin") in small samples of intima is described. Tissue samples were subjected to electrophoresis directly from the intima into an antibody-containing gel to remove and measure fibrinogen and other soluble fibrin reactive antigens (FRA). The residual tissue was then exhaustively incubated with plasmin, and the soluble fragments generated from the insoluble "fibrin" were measured by quantitative immunoelectrophoresis. "Fibrin" accounted for about 2% of the tissue dry weight in normal intima and the ratio fibrinogen/"fibrin" was 1-1.5. In the gelatinous lesions, which seem to be the precursors of fibrous plaques, there was a small increase in "fibrin" but a substantial increase in fibrinogen and low density (LD)-lipoprotein, and the ratio fibrinogen/"fibrin" rose to about 3, which suggests that the increase in "fibrin" is secondary to increased permeation of fibrinogen. At the edges of large plaques there was also a threefold increase in fibrinogen, but "fibrin" increased fivefold, and accounted for 10% of the tissue dry weight. The same high concentration was found in the centres of large fibrous plaques with advanced atheroma lipid. Raised levels of "fibrin" were accompanied by raised levels of fibrinogen in most tissue samples. About 80% of the total soluble FRA could be clotted with thrombin; there was no significant difference between normal intima and lesions, and the proportion clotted was not related to "fibrin" content.