Cell turnover in physiological conditions indicates that differentiated or aged cells are naturally reduced by apoptosis and replaced by the division progeny of adult stem cells. The rate of cell turnover depends on each type of tissue or organ 
. For example, intestinal epithelium is restored every 3–5 days in mammals 
, whereas stratum corneum replacement takes about 14 days and 28 days for replacement of the entire epidermis 
. Urothelial cells display a much slower turnover rate of many weeks, and are basically quiescent until injury. Our hypothesis is that the more urine derived cells, including urothelial cells, renal tubule epithelial cells and others can be shed off from the urinary tract system into urine in the taller individuals due to the body size. We found a turnover rate of approximately 6×104
cells in the urinary tract system over 24 hours, depending on the individual's age and height. Cells from older and taller donors shed off more cells into their urine samples. Although a large of experiment samples were not performed in the present study, the preliminary data showed a strong correlation between numbers of shed cells and individual height and age. Among these total numbers of cells, the living cells were about 4×103
cells (13.7% of the total cells in urine). We used urine from healthy men, since voided urine from women is more likely to be contaminated with epithelial cells or vaginal bacteria. In addition, more cells may be present in urine collected by urethral catheter due to catheter induced injury of the bladder and urethral mucosa. Therefore, it is easier to collect the urine sample and derive more representative cells when cells are collected from voided urine than via a urethral catheter.
There are various sources of autologous mesenchymal stem cells 
, such as bone marrow and adipose tissue. However, our purpose was to identify a cell source with high self-renewal and multi-potent differentiation capacities that can be obtained via a simple and non-invasive approach. We recently found that a subpopulation of cells isolated from voided urine or urine from upper urinary tract 
possess stem cells features, i.e. are highly expandable and have multi-lineage differentiation capability 
. These urine-derived stem cells are capable of multipotent differentiation to mesoderm lineages (i.e. SMCs 
and endothelial cells 
) and endoderm lineages such as urothelial cells 
Our previous studies showed that USCs can survive for only a few hours in urine without any preservation, and eventually all the cells died within 24 hours after the urine was discharged from the body. Urine itself is unfavorable for cell survival due the lack of nutrients, metabolic waste material that might be toxic, osmotic pressure, and a non-physiological pH value. This toxic environment impairs cellular membrane and causes cellular lysis. Two strategies can be used to preserve cells and improve the results of their necessary storage. One is cryopreservation, to spin down the cells from urine and freeze them in dimethyl sulfoxide (DMSO) in liquid nitrogen immediately, which requires the appropriate facilities to isolate and store cells. The other strategy is to add the preservation medium in urine and store whole urine samples at 4°C to retain cell membrane stability, slow down metabolic processes, and prevent cell lysis. The advantage of this approach is that no special equipment or processes are needed to isolate and freeze cells, which could be more convenient for patients.
An effective and simple preservation method is greatly advantageous for accumulating more USCs for necessary storage or for USC distribution within a restricted time period for potential clinical application. When large amounts of USCs at early stages are needed rapidly for cell-based therapy, collecting more urine samples to preserve living USCs is necessary. MSC at earlier passages possess greater plasticity and higher growth potential 
. It would be ideal for cell therapy to obtain USCs at as early passage as possible, since cells often lose differentiation function and proliferation capacity with number of passage in cultures. Our previous studies demonstrated that 5–10 USC colonies can be formed per fresh 100 ml urine. Each single USC clone can generate four million cells at passage 3 within 2 weeks 
, and the USCs at early passage can be more efficiently differentiated into urothelial and smooth muscle cells 
It would be beneficial and convenient for patients to collect their urine samples, refrigerate them at home, and then bring the samples to the hospital rather than keeping them as inpatients. In addition, using urine samples stored for 24 hours allows batching of processing, which minimizes costs and decreases the possibility of contamination in each step of the cell isolation process. The ability to preserve cells in urine permits transportation of the cells if necessary for cell isolation, characterization and culture of USCs for research or future clinical use.
In this study, we optimized preservation techniques to maintain the maximum amount of cell viability and optimal stem cell properties, i.e. self-renewal and multi-potency during cell preservation in urine. In each 100 ml of urine samples, 3–4 USC clones existed in 24-hours preserved urine, 4–5 clones in 12-hours preserved urine, and about 6–7 USC clones in fresh urine. Overall, in about 100 USC clones, nearly 50–80% of fresh USCs can be preserved from adult urine during 24-hours storage by these preservation methods. USCs can be obtained from each donor. Although USCs cannot be obtained from each urine sample, they can be obtained from about 70% of preserved urine specimens of each individual. Importantly, the quality of stored USCs was the same as fresh USCs in each area tested, consistent with our previous study 
. Like fresh USCs, the preserved USC clones initially grew a single rice-grain-like cell and then formed a cluster of 6–9 cells in the initial primary culture. They underwent more than 47 population doublings compared to fresh USCs, with nearly 50 population doublings. The preserved cells expressed mesenchymal stem cell 
and perictye markers 
, such as CD44, CD 73, CD90, and CD105, CD146, but not hematopoietic stem cell markers 
such as CD31, CD34, and CD45. Half (5 of 10) preserved cell clones had high telomerase activity and 4 of 10 fresh USCs had telomerase activity; all had normal karyotypes. Prominently, the preserved USCs maintained bi-potent differentiation capacity. After storage, differentiated USCs expressed myogenic-specific genes and proteins 
such as desmin and myosin contractile function when exposed to myogenic differentiation medium. They also expressed urothelial genes and proteins 
such as uroplakin I and III a, and tight junction genes, proteins such as E-cadherin and cingulin when exposed to urothelial differentiation medium. Furthermore, the urothelially differentiated cells possessed tight junction ultra-microstructure and barrier function.
To optimize preservation conditions, six different solutions were tested, and USC culture medium with serum was the best environment for USCs during simple cold storage, compared to serum alone, culture medium alone or organ preservation solutions. The numbers of the cells declined markedly using other solutions. As the densities of cells and preserved medium (>1) are both slightly higher than urine, the cells sank on the bottle of containers with medium at 4°C during storage due to gravity. Most cells were immersed in the culture medium. This might be why a small amount of preserved medium (about 10–15% of urine volume) can retain the USC clones.
Maintaining stability of the cellular membrane is critical in preservation for stem cells in urine, given its limited content of nutrients and oxygen. Although it is unknown how culture medium with serum provides cell protection, multiple factors might be involved in stabilization of the cell membrane. For example, the culture medium may offer a favorable osmotic concentration, or the presence of serum and supplements in the medium (e.g. hydrocortisone and insulin) may help to maintain cell membrane stability, prevent edema and chemical toxicity 
. Therefore, the combination of culture medium and serum provided better conditions to preserve USC clones. In addition, the lack of glucose in the medium may prevent cellular metabolism. Furthermore, hypothermic storage at 4°C reduces the rate of cell metabolism and oxygen consumption, and thus reduces cellular impairment.
Compared to organ solutions, culture medium provided better conditions for cell preservation in these experiments. Although both HTK Solution 
and University of Wisconsin solution 
have been successfully used in preservation of donor kidneys, livers, pancreas, and hearts, in our studies, neither solution retained USCs better than the USC culture medium. In addition, both media are very expensive and might not be recommended to use in preservation of cells in body fluid.
In summary, human USCs are a potential cell source for cell therapy in the urinary tract system. It would be beneficial to preserve the USCs or cells in urine and also retain cell quality and function of USCs during necessary storage and transportation. In this study, we demonstrated that USCs can be obtained from each individual's preserved urine specimens. The cell quality and function of preserved USCs are the nearly same as the fresh USCs in cell morphology, cell growth patterns, expression of stem cell surface makers, self-renewal capacity, differentiation capacity, expression of telomerase activity, and chromosome stability. Culture media with a minimum of serum added into the urine significantly increased cell viability and maintained membrane integrity of USCs compared with cells in organ preservation solutions. This preservation approach is simple, effective and low cost, which makes it possible to transport living USCs in the urine, or possibly or to preserve cells contained in other body fluids for transportation or for short-term storage.