Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells.

Vukosavljevic B, Hittinger M, Hachmeister H, et al. Vibrational spectroscopic imaging and live cell video microscopy for studying differentiation of primary human alveolar epithelial cells. Journal of Biophotonics. 2019;12(6): e201800052

Coating stability and insertion forces of an alginate-cell-based drug delivery implant system for the inner ear

Long-term drug delivery to the inner ear for neuroprotection might improve the outcome for hearing disabled patients treated with a cochlear implant (CI). Neurotrophic factor (NTF) producing cells encapsulated in an alginate-matrix, to shield them from the host immune system and to avoid migration, and applied as viscose solution or electrode coating could address this requirement. Both application methods were tested for their feasibility in an artificial human cochlea model. Since both strategies potentially influence the electrode implantability, insertion forces and coating stability were analyzed on custom-made electrode arrays. Both, injection of the alginate-cell solution into the model and a manual dip coating of electrode arrays with subsequent insertion into the model were possible. The insertion forces of coated arrays were reduced by 75% of an uncoated reference. In contrast, filling of the model with non-crosslinked alginate-cell solution slightly increased the insertion forces. A good stability of the coating was observed after first insertion (85%) but abrasion increased after multiple insertions (50%). Both application strategies are possible options for cell-induced drug-delivery to the inner ear, but an alginate-cell coating of CI-electrodes has a great potential to combine an endogenous NTF-source with a strong reduction of insertion forces

Stem Cell Based Drug Delivery for Protection of Auditory Neurons in a Guinea Pig Model of Cochlear Implantation

Background: The success of a cochlear implant (CI), which is the standard therapy for patients suffering from severe to profound sensorineural hearing loss, depends on the number and excitability of spiral ganglion neurons (SGNs). Brain-derived neurotrophic factor (BDNF) has a protective effect on SGNs but should be applied chronically to guarantee their lifelong survival. Long-term administration of BDNF could be achieved using genetically modified mesenchymal stem cells (MSCs), but these cells should be protected – by ultra-high viscous (UHV-) alginate (‘alginate-MSCs’) – from the recipient immune system and from uncontrolled migration.Methods: Brain-derived neurotrophic factor-producing MSCs were encapsulated in UHV-alginate. Four experimental groups were investigated using guinea pigs as an animal model. Three of them were systemically deafened and (unilaterally) received one of the following: (I) a CI; (II) an alginate-MSC-coated CI; (III) an injection of alginate-embedded MSCs into the scala tympani followed by CI insertion and alginate polymerization. Group IV was normal hearing, with CI insertion in both ears and a unilateral injection of alginate-MSCs. Using acoustically evoked auditory brainstem response measurements, hearing thresholds were determined before implantation and before sacrificing the animals. Electrode impedance was measured weekly. Four weeks after implantation, the animals were sacrificed and the SGN density and degree of fibrosis were evaluated.Results: The MSCs survived being implanted for 4 weeks in vivo. Neither the alginate-MSC injection nor the coating affected electrode impedance or fibrosis. CI insertion with and without previous alginate injection in normal-hearing animals resulted in increased hearing thresholds within the high-frequency range. Low-frequency hearing loss was additionally observed in the alginate-injected and implanted cochleae, but not in those treated only with a CI. In deafened animals, the alginate-MSC coating of the CI significantly prevented SGN from degeneration, but the injection of alginate-MSCs did not.Conclusion: Brain-derived neurotrophic factor-producing MSCs encapsulated in UHV-alginate prevent SGNs from degeneration in the form of coating on the CI surface, but not in the form of an injection. No increase in fibrosis or impedance was detected. Further research and development aimed at verifying long-term mechanical and biological properties of coated electrodes in vitro and in vivo, in combination with chronic electrical stimulation, is needed before the current concept can be tested in clinical trials

Distributed automated manufacturing of pluripotent stem cell products

Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site–decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing ‘live’ corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow

3D printing of hydrogels in a temperature controlled environment with high spatial resolution

There is great hope in 3D printing techniques to create patient specific scaffolds for therapeutic applications. The majority of these approaches rely on materials that both give support to cells and effectively mimic a tissue specific microenvironment. Hydrogels provide an exceptional support for cells but their physicochemical properties are not suited for conventional additive layer manufacturing. Their low viscosity and resulting fluidic nature inhibit voluminous 3D deposition and lead to crude printing accuracy. To enhance mechanical features, hydrogels are often chemically modified and/or mixed with additives; however it is not clear whether these changes induce effects on cellular behavior or if in vivo applications are at risk. Certainly it increases the complexity of scaffold systems. To circumvent these obstacles, we aimed for a 3D printing technique which is capable of creating scaffolds out of unmodified, pure hydrogels. Here we present a new method to produce alginate scaffolds in a viscosity- independent manner with high spatial resolution. This is achieved by printing in a sub-zero environment which leads to fast freezing of the hydrogels, thus preserving the printed shape and circumventing any viscosity dependent flows. This enables the user to create scaffolds which are able to reflect soft or stiff cell niches

Labbag® - a versatile bag-based cultivation system for expansion, differentiation and cryopreservation of human stem cells

Novel approaches in regenerative medicine and tissue engineering are highly promoted by human induced pluripotent stem cells. Typical workflows include the expansion, differentiation and/or cryopreservation of these cells. Often, the differentiation, cytotoxicity tests or disease modelling rely on multicellular aggregates. Here, we present the novel bag-based system “Labbag®” to address workflows required for these applications. As a unique feature, the Labbag® provides a chemical spot pattern to allow the formation of several hundreds of hanging droplets within a few seconds with standard laboratory equipment. In this study, we describe the general concept of the Labbag® and show aggregate formation of stem cells as first stem cell workflow successfully transferred

Stem Cell Based Drug Delivery for Protection of Auditory Neurons in a Guinea Pig Model of Cochlear Implantation

Background: The success of a cochlear implant (CI), which is the standard therapy for patients suffering from severe to profound sensorineural hearing loss, depends on the number and excitability of spiral ganglion neurons (SGNs). Brain-derived neurotrophic factor (BDNF) has a protective effect on SGNs but should be applied chronically to guarantee their lifelong survival. Long-term administration of BDNF could be achieved using genetically modified mesenchymal stem cells (MSCs), but these cells should be protected – by ultra-high viscous (UHV-) alginate (‘alginate-MSCs’) – from the recipient immune system and from uncontrolled migration. Methods: Brain-derived neurotrophic factor-producing MSCs were encapsulated in UHV-alginate. Four experimental groups were investigated using guinea pigs as an animal model. Three of them were systemically deafened and (unilaterally) received one of the following: (I) a CI; (II) an alginate-MSC-coated CI; (III) an injection of alginate-embedded MSCs into the scala tympani followed by CI insertion and alginate polymerization. Group IV was normal hearing, with CI insertion in both ears and a unilateral injection of alginate-MSCs. Using acoustically evoked auditory brainstem response measurements, hearing thresholds were determined before implantation and before sacrificing the animals. Electrode impedance was measured weekly. Four weeks after implantation, the animals were sacrificed and the SGN density and degree of fibrosis were evaluated. Results: The MSCs survived being implanted for 4 weeks in vivo. Neither the alginate-MSC injection nor the coating affected electrode impedance or fibrosis. CI insertion with and without previous alginate injection in normal-hearing animals resulted in increased hearing thresholds within the high-frequency range. Low-frequency hearing loss was additionally observed in the alginate-injected and implanted cochleae, but not in those treated only with a CI. In deafened animals, the alginate-MSC coating of the CI significantly prevented SGN from degeneration, but the injection of alginate-MSCs did not. Conclusion: Brain-derived neurotrophic factor-producing MSCs encapsulated in UHV-alginate prevent SGNs from degeneration in the form of coating on the CI surface, but not in the form of an injection. No increase in fibrosis or impedance was detected. Further research and development aimed at verifying long-term mechanical and biological properties of coated electrodes in vitro and in vivo, in combination with chronic electrical stimulation, is needed before the current concept can be tested in clinical trials

Global epidemiology of pediatric traumatic spine injury: A systematic review and meta-analysis

Objective: Traumatic spine injury (TSI) leads to significant morbidity and mortality in children. However, the global epidemiology of pediatric TSI is currently unknown. We conducted a systematic review and meta-analysis to estimate the global incidence of pediatric TSI and burden of cases.Methods: PubMed, Embase, and Scopus were searched for articles in June 2021 and updated in March 2023 with no restriction on language and year of publication. A meta-analysis was conducted to estimate the global incidence of pediatric traumatic spine injury (TSI) and subsequently the number of cases of pediatric TSI worldwide and proportion requiring spine surgery.Results: Of 6,557 articles, 25 studies met the inclusion criteria. Road traffic accidents (64%) were responsible for the majority of cases reported in the literature followed by falls (18%). The global incidence of TSI in children ≤ 20 years old was estimated to be 14.24/100000 amounting to 375,734, with an estimated 114,975 requiring spine surgery. Across World Bank income classification groups, lower MICs had the highest pediatric TSI case burden (186,886 cases with 57,187 requiring spine surgery). Across WHO Regions, countries in the Southeast Asia Region (SEAR) had the largest number of projected cases at 88,566 with 27,101 requiring surgical management, followed closely by the African Region (AFR) with 87,235 projected cases with 26,694 requiring surgical management.Conclusion: Pediatric TSI represents a large healthcare burden globally. Interventions targeting both injury prevention and neurosurgical capacity strengthening, especially in low resource settings, are needed to address this global health challenge

Supplementary Information Files for 'Distributed automated manufacturing of pluripotent stem cell products'

Supplementary Information Files for 'Distributed automated manufacturing of pluripotent stem cell products'Abstract:Establishing how to effectively manufacture cell therapies is an industry-level problem. Decentralised manufacturing is of increasing importance, and its challenges are recognised by healthcare regulators with deviations and comparability issues receiving specific attention from them. This paper is the first to report the deviations and other risks encountered when implementing the expansion of human pluripotent stem cells (hPSCs) in an automated three international site–decentralised manufacturing setting. An experimental demonstrator project expanded a human embryonal carcinoma cell line (2102Ep) at three development sites in France, Germany and the UK using the CompacT SelecT (Sartorius Stedim, Royston, UK) automated cell culture platform. Anticipated variations between sites spanned material input, features of the process itself and production system details including different quality management systems and personnel. Where possible, these were pre-addressed by implementing strategies including standardisation, cell bank mycoplasma testing and specific engineering and process improvements. However, despite such measures, unexpected deviations occurred between sites including software incompatibility and machine/process errors together with uncharacteristic contaminations. Many only became apparent during process proving or during the process run. Further, parameters including growth rate and viability discrepancies could only be determined post-run, preventing ‘live’ corrective measures. The work confirms the critical nature of approaches usually taken in Good Manufacturing Practice (GMP) manufacturing settings and especially emphasises the requirement for monitoring steps to be included within the production system. Real-time process monitoring coupled with carefully structured quality systems is essential for multiple site working including clarity of decision-making roles. Additionally, an over-reliance upon post-process visual microscopic comparisons has major limitations; it is difficult for non-experts to detect deleterious culture changes and such detection is slow.</div