ABSTRACT
Advanced three dimensional cell culture techniques have been adopted in many laboratories to better model in vivo tissue by recapitulating multi-cellular architecture and the presence of extracellular matrix features. We describe here a 3D cell culture platform in a small molecule screening workflow that uses traditional biomarker and intracellular kinase end point assay readouts. By combining the high throughput bioprinter RASTRUM with the high throughput screening assay AlphaLISA, we demonstrate the utility of the protocol in 3D synthetic hydrogel cultures with breast cancer (MDA-MB-231 and MCF-7) and fibroblast cells. To establish and validate the workflow, we treated the breast cancer cultures with doxorubicin, while fibroblast cultures were stimulated with the pro-inflammatory lipopolysaccharide. 3D and 2D MDA-MB-231 cultures were equally susceptible to doxorubicin treatment, while showing opposite ERK phosphorylation changes. Doxorubicin readily entered embedded MCF-7 spheroids and markedly reduced intracellular GSK3ß phosphorylation. Furthermore, quantifying extracellular interleukin 6 levels showed a very similar activation profile for fibroblasts in 2D and 3D cultures, with 3D fibroblast networks being more resistant against the immune challenge. Through these validation experiments we demonstrate the full compatibility of the bioprinted 3D cell cultures with several widely-used 2D culture assays. The efficiency of the workflow, minimal culture handling, and applicability of traditional screening assays, demonstrates that advanced encapsulated 3D cell cultures can be used in 2D cell culture screening workflows, while providing a more holistic view on cell biology to increase the predictability to in vivo drug response.
Subject(s)
Bioprinting , Breast Neoplasms , Breast Neoplasms/drug therapy , Cell Culture Techniques, Three Dimensional , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Drug Discovery/methods , Female , Humans , Spheroids, Cellular , WorkflowABSTRACT
A defining feature of tetrapod evolutionary origins is the transition from fish fins to tetrapod limbs. A major change during this transition is the appearance of the autopod (hands, feet), which comprises two distinct regions, the wrist/ankle and the digits. When the autopod first appeared in Late Devonian fossil tetrapods, it was incomplete: digits evolved before the full complement of wrist/ankle bones. Early tetrapod wrists/ankles, including those with a full complement of bones, also show a sharp pattern discontinuity between proximal elements and distal elements. This suggests the presence of a discontinuity in the proximal-distal sequence of development. Such a discontinuity occurs in living urodeles, where digits form before completion of the wrist/ankle, implying developmental independence of the digits from wrist/ankle elements. We have observed comparable independent development of pectoral fin radials in the lungfish Neoceratodus (Osteichthyes: Sarcopterygii), relative to homologues of the tetrapod limb and proximal wrist elements in the main fin axis. Moreover, in the Neoceratodus fin, expression of Hoxd13 closely matches late expression patterns observed in the tetrapod autopod. This evidence suggests that Neoceratodus fin radials and tetrapod digits may be patterned by shared mechanisms distinct from those patterning the proximal fin/limb elements, and in that sense are homologous. The presence of independently developing radials in the distal part of the pectoral (and pelvic) fin may be a general feature of the Sarcopterygii.
Subject(s)
Fishes/anatomy & histology , Fishes/growth & development , Animals , Fishes/genetics , Forelimb/anatomy & histology , Forelimb/growth & development , Fossils , Gene Expression Regulation , PhylogenyABSTRACT
Differentiation of the axial skeleton into distinct regions, once thought to be characteristic of the Tetrapoda, also occurs in the actinopterygian Danio rerio. In these taxa, the boundary between the cervical-thoracic regions correlates with Hoxc6 expression and morphological features such as position of the pectoral fin and associated nerves, and the absence of ribs. In the lungfish Neoceratodus, a member of the extant sister taxon to the Tetrapoda, the first vertebral element to chondrify is situated well posterior to the skull, developing from somites 6 and 7 (6/7) and associated with an enlarged cranial rib and nerves innervating the pectoral fin. Two vertebral elements develop later and more anteriorly, associated with somites 4/5 and 5/6. These three elements become incorporated into the occipital region of the skull during Neoceratodus ontogeny, until the cranial rib itself articulates to the rear of the skull. These features of early development indicate a regionalization of the Neoceratodus vertebral column: the cranial rib marks the boundary between the cervical and thoracic regions, the two more anterior vertebrae lacking ribs represent the cervical region, while somites 1-4 (cranial half), lacking any vertebral development, represent the occipital region. However, the cervical region of the vertebral column is effectively lost during ontogeny of Neoceratodus. A recognizable cervical region in the tetrapod vertebral column, as in zebrafish, suggests that cervical vertebrae are not incorporated into the skull but maintained as distinct elements of the column, representing an important shift in relative developmental timing and the influence of heterochrony in this region during the fish-tetrapod transition.
Subject(s)
Body Patterning/physiology , Cervical Vertebrae/embryology , Fishes/anatomy & histology , Fishes/embryology , Somites/physiology , Animals , Cervical Vertebrae/anatomy & histology , Histological Techniques , Phylogeny , Species Specificity , Video RecordingABSTRACT
This work presents characterisation of deiodinase type III (D3) mRNA as cDNA and the tissue distribution of D3 mRNA in the Australian lungfish, Neoceratodus forsteri. We have identified the full length of a approximately 1.4 kb D3 mRNA in the liver, which has a single in-frame UGA codon and a selenocysteine insertion sequence (SECIS) form 2 in the 3'-UTR. Lungfish D3 mRNA was expressed in all tested tissues (liver, lung, kidney, brain, heart, and gills) as demonstrated by Northern blot analyses. PCR conducted on genomic DNA indicated that the lungfish D3 is a single exon gene. Also, we present enzymatic characteristics of this exclusively IRD enzyme, have determined its substrate preference, DTT cofactor requirements, PTU inhibition, and kinetic properties. These results indicate that lungfish D3 has the typical enzymatic characteristics of vertebrate D3 enzymes.
Subject(s)
Fishes/metabolism , Iodide Peroxidase/analysis , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Brain/enzymology , DNA, Complementary/analysis , Gills/enzymology , Iodide Peroxidase/genetics , Kidney/enzymology , Liver/enzymology , Lung/enzymology , Microsomes, Liver/enzymology , Molecular Sequence Data , Myocardium/enzymology , Organ Specificity , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence AlignmentABSTRACT
Deiodinase type II metabolises the prohormone T4 (thyroxine) into the biologically active hormone T3 (3,5,3'-triiodothyronine), at the cellular level in extrathyroidal target tissues. In juvenile lungfish, Neoceratodus forsteri, a typical deiodinase type II is present in most tissues. We have identified the full length of a 1.8 kb deiodinase type II mRNA in liver, and a truncated (1.3 kb) version in brain. Both mRNAs have two in frame UGA codons, but only the liver form has a predicted SECIS structure (form 1) in its 3'-UTR. We also report the presence of additional different length transcripts of deiodinase II mRNA, i.e., 3, 4, and 8 kb, in liver, and 8 kb in kidney, heart, and gill tissues. Expression of the longer (approximately 8 kb) transcript is very low. Real-time PCR confirmed the low expression of transcripts in all tissues, suggested by the Northern blot analysis.