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David Schaffer

Publications and source records attributed to David Schaffer.

7 recordsLinked to original sources
PubMed2004

NanoLiterBioReactor: long-term mammalian cell culture at nanofabricated scale.

There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10-100 nL volume), as well as cell seeding. Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO(2)/air supply was necessary. The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One is termed "circular NBR with a central post (CP-NBR)," another, "perfusion (grid) NBR (PG-NBR)," and a third version, "multitrap (cage) NBR (MT-NBR)," the last two providing total cell retention. Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design. To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1-5 days following the seeding. Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts. High viabilities were achieved in all designs. We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment. The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature.

Animals↗
PubMed2002

Adeno-associated virus effectively mediates conditional gene modification in the brain.

The Cre/loxP system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that in vivo modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates beta-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.

Animals↗
PubMed2002

Size-dependent intracellular immunotargeting of therapeutic cargoes into endothelial cells.

Cell-selective intracellular targeting is a key element of more specific and safe enzyme, toxin, and gene therapies. Endothelium poorly internalizes certain candidate carriers for vascular immunotargeting, such as antibodies to platelet endothelial cell adhesion molecule 1 (PECAM-1). Conjugation of poorly internalizable antibodies with streptavidin (SA) facilitates the intracellular uptake. Although both small and large (100-nm versus 1000-nm diameter) anti-PECAM/SA-beta galactosidase (SA-beta-gal) conjugates bound selectively to PECAM-expressing cells, only small conjugates showed intracellular accumulation of active beta-gal. To study whether size of the conjugates controls the uptake, a series of anti-PECAM/SA and anti-PECAM/bead conjugates ranging from 80 nm to 5 microm in diameter were produced. Human umbilical vein endothelial cells and PECAM-transfected mesothelioma cells internalized 80- to 350-nm anti-PECAM conjugates, but not conjugates larger than 500 nm. Further, size controls intracellular targeting of active therapeutic cargoes in vitro and in vivo. Small anti-PECAM/DNA conjugates transfected target cells in culture 5-fold more effectively than their large counterpart (350- versus 4200-nm diameter). To evaluate the practical significance of the size-controlled subcellular addressing, we coupled glucose oxidase (GOX) to anti-PECAM and antithrombomodulin. Both types of conjugates had equally high pulmonary uptake after intravenous injection in mice, yet only small (200- to 250-nm), not large (600- to 700-nm), GOX conjugates caused profound oxidative vascular injury in the lungs, presumably owing to intracellular generation of H(2)O(2). Thus, engineering of affinity carriers of specific size permits intracellular delivery of active cargoes to endothelium in vitro and in vivo, a paradigm useful for the targeting of drugs, genes, and toxins.

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PubMed2002

Evidence-based screening criteria for retinopathy of prematurity: natural history data from the CRYO-ROP and LIGHT-ROP studies.

BACKGROUND: The Multicenter Trial of Cryotherapy for Retinopathy of Prematurity (CRYO-ROP) demonstrated the efficacy of treatment for threshold ROP and indicated the need for worldwide ROP screening. Previous guidelines for ROP screening have been largely based on clinical impression; we can now develop evidence-based screening recommendations. OBJECTIVE: To define the appropriate ages and retinal ophthalmoscopic signs that determine when to commence and conclude acute phase ROP screening. DESIGN: Analysis of data from 2 prospective randomized controlled trials: CRYO-ROP (January 1, 1986, to November 30, 1987) and Light Reduction in ROP (LIGHT-ROP) (July 1, 1995, to March 31, 1997). SETTING: Neonatal intensive care units in 23 study centers in the United States for CRYO-ROP and 3 centers for LIGHT-ROP. PATIENTS: Eyes were examined sequentially in 4099 infants with birth weight less than 1251 g (CRYO-ROP study) and in 361 infants with birth weight less than 1251 g and gestational age less than 31 weeks (LIGHT-ROP study). RESULTS: In 99% of infants, retinal conditions indicating a risk of poor outcome were not observed before 31 weeks' postmenstrual age or 4 weeks' chronologic age. Signs indicating that the risk of visual loss from ROP was minimal or had passed were the infant's attainment of 45 weeks' postmenstrual age without the development of prethreshold ROP or worse, progression of retinal vascularization into zone III without previous zone II ROP, and full vascularization. CONCLUSIONS: The initial eye examination should be conducted by 31 weeks' postmenstrual age or 4 weeks' chronologic age, whichever is later. Acute phase ROP screening can be discontinued when any of the 3 signs is present, indicating that the risk of visual loss from ROP is minimal or passed.

Age Factors↗
PubMed2002

Targeted retrograde gene delivery for neuronal protection.

The cellular heterogeneity and complex circuitry of the central nervous system make it difficult to achieve precise delivery of experimental and therapeutic agents. We report here an in vivo retrograde gene delivery strategy to target mature projection neurons using adeno-associated virus, a vector with low toxicity and the capacity for long-term gene expression. Viral delivery to axon terminal fields in the hippocampus and striatum resulted in viral internalization, retrograde transport, and transgene expression in specific projection neurons in entorhinal cortex and substantia nigra. Retrograde delivery of the anti-apoptotic gene Bcl2l (also known as Bcl-xL) protected entorhinal projection neurons from subsequent damage-induced cell death. Given the broad distribution of neurons affected by neurodegenerative diseases, gene delivery to both the terminal fields and the projection neurons through retrograde infection provides for strategic therapeutic intervention at both levels of the neural circuit. This approach may also facilitate experimental studies of defined neural circuits.

Animals↗
PubMed2002

Engineering the surface properties of synthetic gene delivery vectors.

Synthetic gene delivery vehicles are a highly promising approach to gene delivery; however, several problems must still be overcome before they can begin to enjoy dinical success. A number of these problems can be addressed by engineering and optimizing the properties of the vector surface, the component of the particle that interacts and "communicates" with tissues and cells during the delivery process. Surfaces must be engineered to satisfy two ostensibly conflicting constraints: the ability to interact specifically with a target cell while avoiding nonspecific protein interactions, particularly with components of the immune system. We summarize progress that has been made in both these areas and discuss several approaches where the intersection of biological and chemical solutions promises to significantly advance the engineering of synthetic vehicles.

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