Advances and challenges in umbilical cord blood and tissue bioprocessing: procurement and storage

Umbilical cord tissue and blood is banked to complement the rapidly advancing field of tissue engineering and regenerative medicine for both autologous and allogeneic therapeutic applications. Whilst many problems concerning the use of the hematopoietic and multipotential mesenchymal stromal cells contained therein may be addressed through the future development of GMP-compliant manufacturing strategies, collection and bioprocessing of these tissues can be optimised in the present to maximise clinical outcomes. In this review, we describe current procurement, processing and storage approaches for umbilical cord blood and tissue; current challenges and how these may be met to augment translation and use of therapeutics harnessing their derivatives

Mixing theory for culture and harvest in bioreactors of human mesenchymal stem cells on microcarriers

The use of human mesenchymal stem cells (hMSCs) in regenerative medicine is a potential major advance for the treatment of many medical conditions, especially with the use of allogeneic therapies where the cells from a single donor can be used to treat ailments in many patients. Such cells must be grown attached to surfaces and for large scale production, it is shown that stirred bioreactors containing ~200 μm particles (microcarriers) can provide such a surface. It is also shown that the just suspended condition, agitator speed NJS, provides a satisfactory condition for cell growth by minimizing the specific energy dissipation rate, εT, in the bioreactor whilst still meeting the oxygen demand of the cells. For the cells to be used for therapeutic purposes, they must be detached from the microcarriers before being cryopreserved. A strategy based on a short period (~7 min) of very high εT, based on theories of secondary nucleation, is effective at removing >99% cells. Once removed, the cells are smaller than the Kolmogorov scale of turbulence and hence not damaged. This approach is shown to be successful for culture and detachment in 4 types of stirred bioreactors from 15 mL to 5 L

Scale-up of an intensified bioprocess for the expansion of bovine adipose-derived stem cells (bASCs) in stirred tank bioreactors

Cultivated meat is an emerging field, aiming to establish the production of animal tissue for human consumption in an in vitro environment, eliminating the need to raise and slaughter animals for their meat. To realise this, the expansion of primary cells in a bioreactor is needed to achieve the high cell numbers required. The aim of this study was to develop a scalable, microcarrier based, intensified bioprocess for the expansion of bovine adipose-derived stem cells as precursors of fat and muscle tissue. The intensified bioprocess development was carried out initially in spinner flasks of different sizes and then translated to fully controlled litre scale benchtop bioreactors. Bioprocess intensification was achieved by utilising the previously demonstrated bead-to-bead transfer phenomenon and through the combined addition of microcarrier and medium to double the existing surface area and working volume in the bioreactor. Choosing the optimal time point for the additions was critical in enhancing the cell expansion. A significant fold increase of 114.19 ± 1.07 was obtained at the litre scale in the intensified bioprocess compared to the baseline (**p < .005). The quality of the cells was evaluated pre- and post-expansion and the cells were found to maintain their phenotype and differentiation capacity

Needle to needle robot-assisted manufacture of cell therapy products

Advanced therapeutic medicinal products (ATMPs) have emerged as novel therapies for untreatable diseases, generating the need for large volumes of high-quality, clinically-compliant GMP cells to replace costly, high-risk and limited scale manual expansion processes. We present the design of a fully automated, robot-assisted platform incorporating the use of multiliter stirred tank bioreactors for scalable production of adherent human stem cells. The design addresses a needle-to-needle closed process incorporating automated bone marrow collection, cell isolation, expansion, and collection into cryovials for patient delivery. AUTOSTEM, a modular, adaptable, fully closed system ensures no direct operator interaction with biological material; all commands are performed through a graphic interface. Seeding of source material, process monitoring, feeding, sampling, harvesting and cryopreservation are automated within the closed platform, comprising two clean room levels enabling both open and closed processes. A bioprocess based on human MSCs expanded on microcarriers was used for proof of concept. Utilizing equivalent culture parameters, the AUTOSTEM robot-assisted platform successfully performed cell expansion at the liter scale, generating results comparable to manual production, while maintaining cell quality postprocessing

Dendritic Cells and Hepatocytes Use Distinct Pathways to Process Protective Antigen from Plasmodium in vivo

Malaria-protective CD8+ T cells specific for the circumsporozoite (CS) protein are primed by dendritic cells (DCs) after sporozoite injection by infected mosquitoes. The primed cells then eliminate parasite liver stages after recognizing the CS epitopes presented by hepatocytes. To define the in vivo processing of CS by DCs and hepatocytes, we generated parasites carrying a mutant CS protein containing the H-2Kb epitope SIINFEKL, and evaluated the T cell response using transgenic and mutant mice. We determined that in both DCs and hepatocytes CS epitopes must reach the cytosol and use the TAP transporters to access the ER. Furthermore, we used endosomal mutant (3d) and cytochrome c treated mice to address the role of cross-presentation in the priming and effector phases of the T cell response. We determined that in DCs, CS is cross-presented via endosomes while, conversely, in hepatocytes protein must be secreted directly into the cytosol. This suggests that the main targets of protective CD8+ T cells are parasite proteins exported to the hepatocyte cytosol. Surprisingly, however, secretion of the CS protein into hepatocytes was not dependent upon parasite-export (Pexel/VTS) motifs in this protein. Together, these results indicate that the presentation of epitopes to CD8+ T cells follows distinct pathways in DCs when the immune response is induced and in hepatocytes during the effector phase

The importance of alternative host plants as reservoirs of the cotton leaf hopper, Amrasca devastans, and its natural enemies

Many agricultural pests can be harboured by alternative host plants but these can also harbour the pests’ natural enemies. We evaluated the capacity of non-cotton plant species (both naturally growing and cultivated) to function as alternative hosts for the cotton leaf hopper Amrasca devastans (Homoptera: Ciccadellidae) and its natural enemies. Forty-eight species harboured A. devastans. Twenty-four species were true breeding hosts, bearing both nymphal and adult A. devastans, the rest were incidental hosts. The crop Ricinus communis and the vegetables Abelmoschus esculentus and Solanum melongena had the highest potential for harbouring A. devastans and carrying it over into the seedling cotton crop. Natural enemies found on true alternative host plants were spiders, predatory insects (Chrysoperla carnea, Coccinellids, Orius spp. and Geocoris spp.) and two species of egg parasitoids (Arescon enocki and Anagrus sp.). Predators were found on 23 species of alternative host plants, especially R. communis. Parasitoids emerged from one crop species (R. communis) and three vegetable species; with 39 % of A. devastans parasitised. We conclude that the presence of alternative host plants provides both advantages and disadvantages to the cotton agro-ecosystem because they are a source of both natural enemy and pest species. To reduce damage by A. devastans, we recommend that weeds that harbour the pest should be removed, that cotton cultivation with R. communis, A. esculentus, and S. melongena should be avoided, that pesticides should be applied sparingly to cultivate alternative host plants and that cotton crops should be sown earlier

Genetički polimorfizmi u dijabetesu: Utjecaj na terapiju oralnim antidijabeticima

Due to new genetic insights, etiologic classification of diabetes is under constant scrutiny. Hundreds, or even thousands, of genes are linked with type 2 diabetes. Three common variants (Lys23 of KCNJ11, Pro12 of PPARG, and the T allele at rs7903146 of TCF7L2) have been shown to be predisposed to type 2 diabetes mellitus across many large studies. Individually, each of these polymorphisms is only moderately predisposed to type 2 diabetes. On the other hand, monogenic forms of diabetes such as MODY and neonatal diabetes are characterized by unique clinical features and the possibility of applying a tailored treatment. Genetic polymorphisms in drug-metabolizing enzymes, transporters, receptors, and other drug targets have been linked to interindividual differences in the efficacy and toxicity of a number of medications. Mutations in genes important in drug absorption, distribution, metabolism and excretion (ADME) play a critical role in pharmacogenetics of diabetes. There are currently five major classes of oral pharmacological agents available to treat type 2 diabetes: sulfonylureas, meglitinides, metformin (a biguanide), thiazolidinediones, and α-glucosidase inhibitors. Other classes are also mentioned in literature. In this work, different types of genetic mutations (mutations of the gene for glucokinase, HNF 1, HNF1ß and Kir6.2 and SUR1 subunit of KATP channel, PPAR-γ, OCT1 and OCT2, cytochromes, direct drug-receptor (KCNJ11), as well as the factors that influence the development of the disease (TCF7L2) and variants of genes that lead to hepatosteatosis caused by thiazolidinediones) and their influence on the response to therapy with oral antidiabetics will be reviewed.Dijabetes tipa 2 dosegao je proporcije epidemije u SAD (> 18 milijuna) i cijelom svijetu (170 milijuna oboljelih osoba) te ima tendenciju daljnjeg dramatičnog rasta. Stoga se u posljednje vrijeme ulažu napori da se otkriju i razviju novi farmakološki agensi za liječenje ove bolesti. Klasifikacija šećerne bolesti proširena je uspjesima istraživača na području genetike. Da bismo razumjeli farmakogenetiku antidijabetika neophodno je razumjeti genetiku samog dijabetesa. Kao što će biti prikazano u ovom radu veliki broj gena koji su povezani s razvojem dijabetesa takođe utječu i na odgovor na terapiju antidijabeticima. S druge strane, mutacije gena koji utječu na ADME (apsorpcija, distribucija, metabolizam i ekskrecija) lijeka imaju značajan utjecaj na farmakogenetiku oralnih antidijabetika. Utvrđeno je da je dijabetes genetički heterogena bolest. Uobičajeni oblici dijabetesa su gotovo uvijek poligenski i za razvoj same bolesti vrlo su značajne snažne interakcije među različitim genima kao i između gena i okoliša. Zbog toga mutacije ili polimorfizmi koji u manjoj mjeri utječu na funkciju gena mogu postati klinički značajni samo u slučaju kada se kombiniraju s drugim faktorima odnosno genima. Smatra se da u razvoju dijabetesa mogu sudjelovati stotine pa čak i tisuće gena. Do 2006. identificirano je nekoliko uobičajenih alela koji povećavaju rizik za razvoj dijabetesa, od kojih su najznačajniji PPARG (Pro12), KCNJ11 (Lys23) i TCF7L2 (T na rs7903146). Do danas je najveći uspjeh postignut u identifikaciji gena odgovornih za razmjerno rijetke oblike ove bolesti poput ”Maturity-onset diabetes of the young” (MODY) i neonatalnog dijabetesa. Monogenske oblike dijabetesa odlikuju jedinstvene kliničke karakteristike i mogućnost primjene individualnog tretmana. Genetički polimorfizmi enzima koji utječu na metabolizam lijekova, transportera, receptora i drugih ciljeva djelovanja lijekova povezani su s interindividualnim razlikama u efikasnosti i toksičnosti mnogih lijekova. Vrlo je važno da se na temelju farmakogenetičkih istraživanja mogu predvidjeti neki neželjeni efekti lijekova. Trenutačno postoji pet glavnih klasa oralnih antidijabetika: sulfoniluree, meglitinidi, metformin (bigvanid), tiazolidindioni i inhibitori α-glukozidaze. U literaturi se također spominju inhibitori dipeptidil peptidaze IV (DPP-IV), selektivni antagonisti kanabinoidnog receptora 1 (CB-1), glukagonu slični peptid 1 mimetici i amilin mimetici. Razumijevanje mehanizama koji rezultiraju disfunkcijom β stanica na fiziološkom i molekularnom nivou neophodno je za napredak u razumijevanju tretmana dijabetesa. U ovom radu dat je pregled različitih genetičkih mutacija (mutacije gena za glukokinazu, HNF 1, HNF1ß, Kir6.2 i SUR 1 podjedinicu KATP kanala ß stanica, PPAR-γ, OCT1 i OCT2, citohrome, KCNJ11, faktore koji utječu na razvoj bolesti (TCF7L2) i varijante gena koji dovode do hepatosteatoze uzrokovane tiazolidindionima) te njihov utjecaj na odgovor na terapiju oralnim antidijabeticima

Assessment of variation in immunosuppressive pathway genes reveals TGFBR2 to be associated with prognosis of estrogen receptor-negative breast cancer after chemotherapy

Introduction: Tumor lymphocyte infiltration is associated with clinical response to chemotherapy in estrogen receptor (ER) negative breast cancer. To identify variants in immunosuppressive pathway genes associated with prognosis after adjuvant chemotherapy for ER-negative patients, we studied stage I-III invasive breast cancer patients of European ancestry, including 9,334 ER-positive (3,151 treated with chemotherapy) and 2,334 ER-negative patients (1,499 treated with chemotherapy). Methods: We pooled data from sixteen studies from the Breast Cancer Association Consortium (BCAC), and employed two independent studies for replications. Overall 3,610 single nucleotide polymorphisms (SNPs) in 133 genes were genotyped as part of the Collaborative Oncological Gene-environment Study, in which phenotype and clinical data were collected and harmonized. Multivariable Cox proportional hazard regression was used to assess genetic associations with overall survival (OS) and breast