Clinically compliant cryopreservation of differentiated retinal pigment epithelial cells.

BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease. METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location. RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology. CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

Karolinska Institutet Human Embryonic Stem Cell Bank.

The Karolinska Institutet Human Embryonic Stem Cell Bank (KI Stem Cell Bank) was established at KI, Stockholm, Sweden, when the first human embryonic stem cell (hESC) line was derived by Professor Hovatta and colleagues in 2002. Since then, the bank has grown to include 60 hESC lines. From the very beginning the aim of the bank has been derivation of hESC lines suitable for clinical use. Step by step progress has been made towards this goal, including removal of xeno components, establishment of chemically defined conditions and Good Manufacturing Practice (GMP) compliancy. Today our bank includes such clinical grade hESC line, KARO1, derived and banked according to GMP guidelines. Many of the hESC lines in the bank have been distributed to the scientific community and are deposited in the Stockholm Medical Biobank available for research on collaborative basis.

Pandemic influenza A(H1N1)pdm09 seroprevalence in Sweden before and after the pandemic and the vaccination campaign in 2009.

The immunity to pandemic influenza A(H1N1)pdm09 in Sweden before and after the outbreaks in 2009 and 2010 was investigated in a seroepidemiological study. Serum samples were collected at four time points: during 2007 (n = 1968), in October 2009 (n = 2218), in May 2010 (n = 2638) and in May 2011 (n = 2513) and were tested for hemagglutination inhibition (HI) antibodies. In 2007, 4.9% of the population had pre-existing HI titres ≥40, with the highest prevalence (20.0%) in 15-24 year-olds, followed by ≥80 year-olds (9.3%). The overall prevalence of HI titres ≥40 had not changed significantly in October 2009. In May 2010 the prevalence had increased to 48.6% with the highest percentages in 5-14 year-olds (76.2%) andlowest in 75-79 year-olds (18.3%). One year later the prevalence of HI titres ≥40 had increased further to 52.2%. Children 5-14 years had the highest incidence of infection and vaccine uptake as well as the highest post-pandemic protective antibody levels. In contrast, the elderly had high vaccine uptake and low attack rate but low levels of protective antibodies, underlining that factors other than HI antibodies are involved in protection against influenza A(H1N1)pdm09. However, for all age-groups the seroprevalence was stable or increasing between 2010 and 2011, indicating that both vaccine- and infection-induced antibodies were long-lived.

The Eurocine L3 adjuvants with subunit influenza antigens induce protective immunity in mice after intranasal vaccination.

The immunogenicity and protective efficacy in mice of intranasally (i.n.) administrated influenza subunit antigens together with lipid-based adjuvants (Eurocine) were compared to those of subcutaneous (s.c.) immunisation. Influenza hemagglutination inhibition (HAI) and ELISA IgG titers were similar in the group's vaccinated s.c. and after i.n. vaccination with adjuvants. The virus-specific IgA levels in serum were higher after vaccination i.n. with adjuvant than after s.c immunisation. Virus-specific IgA was measurable in nasal washings only after i.n vaccinations, with and without adjuvants. Thus, i.n. vaccination with the endogenous non-toxic, lipid adjuvants induced equal or stronger antibody responses as compared to s.c. immunisation with the same antigen. We further analysed the protective efficacy against virus challenge in a mouse model. A subunit antigen preparation of the A/New/Caledonia/20/99 strain was used for vaccination of NMRI mice with different combinations of adjuvants. The mice were challenged i.n. with 6.5 tissue culture infectious doses(50) of homologous virus and sacrificed 3 days later. Since the virus is not lethal in mice, the protective efficacy was measured by quantitative, real-time PCR on pulmonary tissue, obtained at autopsy. The mice treated with only adjuvant and the group of naïve mice clearly had the highest mean viral RNA copy numbers (19.200 and 11.000, respectively). All vaccinated groups had significantly lower copy numbers, especially the mice that received the L3A i.n. (-median 120; i.n. L3B-median 2.200; and non-adjuvanted s.c. vaccination-median 2.600). Our findings prompt further investigations of the effect of the formulations in ferrets, monkeys and man.