A modified procedure for replica plating of mammalian cells allowing selection of clones based on gene expression.

The polyester cloth replica-plating technique for selection of mammalian cell clones was modified by growing cells in colonies on a flexible polytetrafluoroethylene membrane and then transferring them completely to polyester cloth (27-microns mesh), from which a replica was made by allowing cells to transfer to a cloth of smaller pore size (17-microns mesh). Using this technique, two phenotype selection methods are demonstrated here: in situ hybridization for detection of a specific mRNA and a photographic film assay for detection of luciferase expression. Cells were transfected with pSV2AL-A delta 5' in which firefly luciferase cDNA is under the control of the simian virus 40 promoter. The luciferase assay was adapted for colonies on polyester cloth; cells were permeabilized with digitonin to allow access of ATP and luciferin to the cell without disruption of colonies. Clones selected for expression or nonexpression of luciferase by the photographic film assay were positive or negative for expression after isolation from the cloth replica and subsequent growth under conventional culture conditions. The replica-plating procedure described here should be generally applicable to most mammalian cell types. The ability to produce replicas of colonies, combined with in situ hybridization or assays that can be adapted to in situ detection, provides phenotype selection for clones based on gene expression independent of growth characteristics.

Production of microspheres with surface amino groups from blends of Poly(Lactide-co-glycolide) and Poly(epsilon-CBZ-L-lysine) and use for encapsulation.

Microspheres were formed from blends of the biodegradable polymer poly(DL-lactic-co-glycolic acid) (PLGA) together with poly(epsilon-CBZ-L-lysine) (PCBZL) by a double-emulsification/solvent evaporation technique. The size of the microspheres formed by this method was dependent both on the total concentration of the polymers and on the ratio of PLGA to PCBZL. The use of the microspheres for encapsulation was demonstrated by the inclusion of a solution of Texas Red fluorescent dye. Lysine epsilon-amino groups on the surface of the microspheres were deprotected by acid hydrolysis or lithium/liquid ammonia reduction. Acid hydrolysis damaged the surface of the microspheres as assessed by scanning electron microscopy, whereas deprotection by lithium/ammonia produced less damage and allowed the retention of encapsulated dye solution. The surface lysine groups made available on the surface of the microspheres could be used to covalently link a variety of biologically active molecules to alter their in vivo properties and allow targeting to specific cell types.

Transfection of cells mediated by biodegradable polymer materials with surface-bound polyethyleneimine.

Poly(epsilon-CBZ-L-lysine) can be mixed with biodegradable polymers such as poly(D,L-lactic-co-glycolic acid) or poly(L-lactic acid) and formed into films, foams, or microspheres. Surface amino groups may then be deprotected with acid or lithium/liquid ammonia. The amino groups serve as a method to modify the surface by attachment of other molecules. In the present experiments, we show that these polymer materials, as films or foams, may be surface modified by the attachment of polyethyleneimine (PEI). Plasmid DNA attached to the PEI can transfect cells plated on the surface over several days. Covalent atachment of PEI was required for transfection to be efficient. PEI was also attached to surface-bound collagen on cell culture plates and was shown to mediate transfection.

Transfection by polyethyleneimine-coated microspheres.

UNLABELLED: Polyethyleneimine (PEI) can be used as a DNA delivery mechanism in cell culture and in vivo. Cells can be transfected by using surface-bound PEI, as well as by PEI/DNA microparticles. In the present experiments we extended these observations by preparing microspheres with covalently attached PEI. Blends of poly(epsilon-CBZ-L-lysine) mixed with poly(D,L-lactic-co-glycolic acid) were formed into microspheres using a double-emulsification/solvent evaporation procedure. CBZ (carbobenzoxy) groups on the surface of microspheres were removed by Li(0) /liquid ammonia reduction. Surface amino groups were used for covalent attachment of PEI and other molecules. Silica microspheres with bonded-phase PEI were also used. Microspheres were mixed with plasmid DNA encoding green fluorescent protein and added to cultured cells. PEI-coated microspheres transfected cultured Caco cells and MH-S alveolar macrophages. Expression of the transfected DNA increased over several days. MH-S cells phagocytosed PEI-coated silica microspheres, which were shown to reside in an acidic subcellular compartment. This was demonstrated by conjugating a pH-sensitive fluorescent dye (seminaphthofluorescein, SNAFL) to the microsphere surface. Transfection of MH-S cells was increased when plasmid DNA was complexed with histone on the surface of the microspheres. CONCLUSION: PEI-coated microspheres have potential as a DNA delivery device with advantages of the unique properties of PEI and ease of surface chemical modification.

Modification of materials formed from poly(L-lactic acid) to enable covalent binding of biopolymers: application to high-density three-dimensional cell culture in foams with attached collagen.

We describe a method for increasing the hydrophilicity of materials formed from biodegradable polymers and introducing chemical functional groups on their surfaces. Poly(L-lactic acid) was blended with poly(epsilon-CBZ-L-lysine) at an 80:20 ratio. Films of the mixture were prepared and foams were made by solvent casting and salt leaching. Amino groups on the surface of the polymer mixture were deprotected by acid hydrolysis. As an example of the applicability of the technique for attachment of biomolecules, we covalently linked collagen to the deprotected amino groups, creating a surface capable of high density growth of a differentiated cell type (bovine adrenocortical cells). The method should be generally useful for surface modification of biodegradable polymer materials used in tissue engineering.

Cutaneous window for in vivo observations of organs and angiogenesis.

BACKGROUND AND OBJECTIVE: The continuous observation in experimental animals of internal organs and processes, such as wound healing and angiogenesis, has been achieved using a variety of transparent windows and chambers. Our objective was to develop procedures for these observations using disposable material for the window and simple surgical techniques. METHODS: For observation of wound healing in the mouse kidney, the kidney was externalized and a wedge was excised. An oval window of polyvinyl chloride film was sutured in place in the skin over the wound. The progress of healing of the wound was observed through the window over 10 days. For observation of angiogenesis, adrenocortical cells were transplanted beneath fascia and muscle and a window was sewn into the skin above the site of transplantation. RESULTS: Clear observations could be made using these cutaneous windows over the period of the experiments. Healing of a wound in the kidney was photographed and measured. The growth of new blood vessels over the site of adrenocortical cell transplantation was observed. CONCLUSIONS: Continuous in vivo observations of organs such as the kidney and processes such as angiogenesis can be made in experimental animals using this simple technique.