BM-MSCs have generated great excitement in the field of regenerative medicine and are being investigated to treat a wide variety of medical conditions. The route of delivery is of essential importance for the outcome of clinical trials using BM-MSCs. While physicians prefer to use the narrowest bore needles for injection in order to reduce patient discomfort and pain as well as to reduce oozing from the injection site, biologists have concerns about the effect of the injection procedure on the viability and biological activity of the cells. The main concerns of cell delivery through very fine needles is that the cells may not survive the shear force, and the larger is the cell size, the more difficult it is for the cells to survive after ejection from the needle. MSCs on average are usually between 8 – 20 microns in size which is significantly larger compared to hematopoietic stem cells or lymphocytes . This raises the concerns among the clinician community that MSCs could be damaged if they are injected via small guage needles for direct subcutaneous or intramuscular delivery into the skin, muscle tissues and vascular organs like heart, liver and spleen or i.v delivery. Our study clearly demonstrates that MSCs could be successfully delivered into these tissues using small guage needles without hampering the biological activity of these cells. Some conditions, including ischemic heart disease and critical limb ischemia, local injection or even multiple injections is required, therefore, the safety of the cells plus needle as a “combined product” causes much concern among regulators and clinical investigators. In this manuscript, we have reported the viability, senescence, surface marker, tri-lineage in vitro differential potential, cryopreservation and in vivo tracking of BM-MSCs which were injected through small gauge needles and those undergoing multiple injections, providing evidence that BM-MSCs are robust and remain fit after needle injection.
Although the clinical applications of cell therapy are still in their infancy, there is an urgent need to determine safe delivery systems where these cells retain their viability and biological functionality . There have been a few recent studies investigating the actual effect of cell suspension passing through a needle based delivery device; for example, Kondziolka and coworkers assessed the viability of neuronal cells passed through a 25 -gauge needle and cannula using a simple trypan blue exclusion method . Heng and co-workers previously reported the effects of injection through 26-Gauge Nitinol needle at different flow rate on MSCs . In the current study we have clearly demonstrated that the BM-MSCs retain their viability and biological function after injecting through different bore size needles.
We also examined the status of 26 G needle injected MSCs after cryopreservation. Though there was a slight drop of cell viability, our data clearly demonstrated that the 26 G needle ejected MSCs were able to maintain their stromal phenotypes and differentiation potential after cryopreservation and subsequent thawing (Figure 3M). Our earlier reports and studies by other research groups have showed that there would be 10 - 20% reduction in post thaw of cell viability [27, 28]. In consistence with these reports, here we have witnessed similar drop of cell viability after cryopreservation and thawing of 26 G needle ejected MSCs.
In certain indications of cell therapy such as the multiple intramuscular injections for critical limb ischemia, repeated intra-muscular injections are made from the same syringe. This study also attempted to mimic this clinical condition as well. We also aimed to find out the clinically relevant smallest possible bore size needles which can be used for safe delivery of cells either by single or multiple injections. We assessed the viability of hMSCs following their ejection through three different clinically relevant bore size needle gauges; 24, 25 and 26 G. The selection of an appropriate and safe needle gauge used during a cell therapy application very much depends on post delivery cellular and functional characteristics, namely the viable cell density, phenotypic expression of the mesenchymal stromal markers, cell senescence and the functional properties such as differentiation of MSCs into mesoderm lineage. As such, cells injected through various needle gauge sizes ranging from 24 to 26 G, have successfully demonstrated their cellular and functional properties. During the second phase of the study, cells were injected through 26 G bore size needle multiple times to mimic certain clinical usage as described above. Cells injected multiple times via 26 gauge bore size needle have also been shown to retain their cellular and functional characteristics.
Since the clinical success of stem cell therapy, in other words restoration of function, tissue integration and/or cell localization  is based upon the post transplantation response, the quality of cells injected are an important determinant of the clinical response. Thus, we were interested to see the in-vivo migration of MSCs after injecting trough the smallest bore size needle. Earlier researchers demonstrated in-vivo migration ability of MSCs [30, 31]; however it was not clear about the needle size used to deliver the cells. Here we have showed that the MSCs injected via 26 G needle were safe and migrated to various organs (Figure 5B-C). It has been shown by other research groups that the MSC will migrate to the thoracic cavity immediately after implantation [32, 33]. In consistency with these studies we also witnessed similar migration pattern where the MSCs immediately migrated to the thoracic cavity after 30 minutes of injection (Figure 5B) and subsequently to the abdominal cavity and towards the lower limbs after 24 hours (Figure 5C) of implantation. These results confirm that cells were healthy and able to migrate all over the body in the normal healthy animal safely after injecting through 26 G needle. Earlier most of the researches applied the IVIV system to identify in-vivo migratory properties of tumor cells in cancer biology. However, here we have successfully demonstrated the usage of IVIS system for studying in-vivo MSC migration pattern.
Conclusion and future perception
Although still in its infancy, cell therapy holds huge promise for the treatment of many diseases with unmet medical needs. It has already been demonstrated by many researchers and clinical trials that the delivery of cells is possible using conventional delivery systems, which is via direct injection in situ. We have clearly demonstrated that there is no significant difference in viability and cellular responses caused by the delivery system after post-ejection via different bore size needles up to a minimum of 26 G. This study also further highlights that multiple injections of cells immediately to the desired site is also safe and non detrimental for the cells. Most clinicians would be comfortable with the use of 26 G needles for most clinical purposes. This makes it possible to use direct needle injection into even vascular organs, thus in future therapies, possible injections to the heart muscle, liver, pancreas or even the spleen would also be safe and non injurious to the cells.