Glass needle-mediated microinjection of macromolecules and transgenes into primary human mesenchymal stem cells

Abstract

Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into various tissue types, including bone, cartilage, tendon, adipocytes, and marrow stroma, making them potentially useful for human cell and gene therapies. Our objective was to demonstrate the utility of glass needle-mediated microinjection as a method to deliver macromolecules (e.g. dextrans, DNA) to hMSCs for cell and molecular biological studies. hMSCs were isolated and cultured using a specific fetal bovine serum, prescreened for its ability to promote cell adherence, proliferation, and osteogenic differentiation. Successful delivery of Oregon Green-dextran via intranuclear microinjection was achieved, yielding a postinjection viability of 76 +/- 13%. Excellent short-term gene expression (63 +/- 11%) was achieved following microinjection of GFP-containing vectors into hMSCs. Higher efficiencies of short-term gene expression ( approximately 5-fold) were observed when injecting supercoiled DNA, pYA721, as compared with the same DNA construct in a linearized form, YA721. Approximately 0.05% of hMSCs injected with pYA721 containing both the GFP and neomycin resistance genes formed GFP-positive, drug-resistant colonies that survived >120 days. Injection of linearized YA721 resulted in 3.6% of injected hMSC forming drug-resistant colonies, none of which expressed GFP that survived 60-120 days. These studies demonstrate that glass needle-mediated microinjection can be used as a method of delivering macromolecules to hMSCs and may prove to be a useful technique for molecular and cell biological mechanistic studies and future genetic modification of hMSCs.

AFM as a tool to probe and manipulate cellular processes

“…In the future, nanotechnology will be applied in cellular biology to influence and control the homeostasis of individual cells in relation to a group of cells and allow the design of complex cell assemblies. Microcapillary techniques are one of the most commonly used methods to introduce and extract genes, proteins, molecules and other materials into and from individual cells [71,72]. However, these techniques are not very accurate in terms of spatial displacement and often cause irreversible damage to sensitive cells.…”

Nanofountain‐Probe‐Based High‐Resolution Patterning and Single‐Cell Injection of Functionalized Nanodiamonds

“…To reduce invasiveness, several methods employing nanoscale tools have been developed. These include nanoneedles,61–64 microcantilevers,65–68 optical nanoinjection,69 and electrochemical attosyringes 70. In the case of nanoneedles and microcantilevers, the material to be injected is immobilized on the exterior surface of the probe.…”

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

“…Microinjection is a common technique for introducing genes into eggs to generate transgenic animals (Brinster et al 1985). It is also used for gene transfer into cells that are difficult to transfect such as vascular smooth muscle cells and mesenchymal stem cells (Nelson & Kent 2002, Tsulaia et al 2003. Furthermore, it has been used to study plasmid DNA degradation, transport, and integration (Folger et al 1982, Dean 1997, Lechardeur et al 1999.…”