AUTOMATION OF SIZING FOR EMBRYONIC STEM CELL AGGREGATES

INTRODUCTION Embryonic stem cells (ESCs) are derived from the inner cell mass of a blastocyst; ESCs are of significant importance to cell and tissue engineering due to their pluripotent status, the ability to form all cell types within the body [1]. Pluripotency is greatly valued in regenerative medicine by providing a potential cell source to restore function to damaged tissues. A large quantity of stem cells is required in order for a stem cell therapy to be successful. Previous works have focused on expanding stem cell populations through stirred suspension bioreactors [2,3]. Bioreactors provide considerable advantages when compared to static tissue culture flask, including ease of automation and monitoring of specific parameters such as oxygen consumption and pH. ESCs cultured in bioreactors grow in cell clumps called aggregates, as seen in Fig 1. These aggregates are sized throughout the culture period as a measure of growth and to assess mass transfer limitations. Commonly used methods to size aggregates are to use microscope images and software to manually determine the longest diameter and the orthogonal diameter. These methods are labour intensive and are susceptible to human error. To address common issues with current methods of aggregate sizing, this project focuses on automating aggregate sizing by developing a plugin for the image analysis software, ImageJ by the US National Institute of Health [4]. METHODS Through development of the ImageJ plugin a process of image filters and enhancements were used to adjust the aggregates within an image so that sizing can occur. Based on Hunt’s use of ImageJ to analyze ESC culture growth and differentiation [5] we were able to determine suitable filters to process our aggregate images. Obtaining orthogonal diameter for each aggregate was accomplished by modifying the particle analyzer in ImageJ. Dougherty’s Measure Roi plugin [6] formed the basis in developing a method to obtain the orthogonal diameter. DISCUSSION AND CONCLUSIONS By developing a plugin for ImageJ to automatically size aggregates we have developed methods to process aggregate images and have extended the particle analyzer to include a measurement for the orthogonal diameter. This allows for automation of the aggregate sizing process.   Future studies should assess the ability of using ImageJ to analyze and accurately size aggregates. The study should consider the application of different sets of image filters to increase accuracy of measurements. A comparison of manual aggregate sizing to automated aggregate sizing should be performed to ensure usability

METABOLISM OF SKIN DERIVED PRECURSOR (SKP) CELLS IN STIRRED SUSPENSION BIOREACTORS

INTRODUCTION More than six million people suffer from burn injuries every year. These injuries can result in psychological trauma, disabilities and permanent disfigurement. A common treatment for burn victims is an autologous graft surgery in which skin is transplanted from a healthy part of the body to the injury site (i.e. split thickness skin graft). This graft, however, does not contain functional dermal tissue, hair follicles or glands, often causing graft contraction, chronic irritation, and unnatural in appearance. We have hypothesized that skin-derived precursor (SKP) cells, a multipotent dermal stem cell that resides within skin hair follicles, can be utilized in conjunction with split thickness skin grafts to improve their function and minimize irritation. SKPs have a high proliferative potential and need to be expanded in a well-controlled, standardized culture environment before they can be utilized in clinical treatments [1]. It is essential to optimize the expansion of SKP cells in order to generate a bioprocess capable of producing enough cells for a clinical setting. METHODS -qNUTR = ∆[NUTR]/Int(Xv)dtThe specific uptake and production rates were calculated for cells cultured in static T-Flask environments and stirred suspension bioreactors run at 40, 60, 80, and 100 rpm. Equation 1 was used to calculate specific rates (qNUTR). The integral change in viable cells (Int(Xv)dt) was calculated using a numerical trapezoid approximation, and the change in nutrient concentration (∆[NUTR]) was measured using the Nova Bioprofile 100+ analyzer. SKP cells were taken from a 68 year old female. RESULTS The specific uptake rates of glucose and glutamine and specific production rates of lactate and ammonia have been determined under altered cell culture environments for SKP cells (Figure1). This provides details into nutrient limitations and cell metabolic behaviours needed to access parameters to guide our bioprocess design and development of robust expansion protocols. DISCUSSION AND CONCLUSIONS Low levels of oxygen and nutrients result in significant changes to cell growth rates [2].  We were able to conclude that SKP cell growth is not limited by the glucose or glutamine concentrations in the media, and lactate and ammonia do not reach toxic levels. It is interesting to note that the stirred suspension environment does appear to have an effect on the specific consumption rates of glucose and glutamine. According to these initial results, we are predicting that under shear stress environments, SKP cells are changing their metabolic behaviour to allow more glucose to convert to pyruvate and enter the TCA cycle. The specific production rates of lactate and ammonia, however, do not follow the same patterns. Further validation and reasons behind these differences need to be investigated

Development of an alternative harvesting method using pH to detach adherent cells from microcarriers

Peripheral nerve injuries are common in Canada, affecting 2.8% of trauma patients treated every year. Current repair strategies are inadequate and repair is often suboptimal with only 25% of patients recovering full motor function and only 3% regaining full sensory function. Because of this, the field is turning toward regenerative medicine to develop a cellular therapy using Schwann cells to repair injured nerves. Schwann cells differentiated from skin derived precursors (SKP-SCs) are a promising cell type as they are easily obtained and allow for autologous therapy. To be able to generate clinically relevant numbers of SKP-SCs, bioreactors need to be used. Since SKP-SCs are an adherent cell type, to be expanded in suspension bioreactors, small spherical beads known as microcarriers need to be used. Our lab has previously shown that these SKP-SCs readily attach to the microcarriers and grow in stirred suspension bioreactors. We have also shown that by controlling the culture parameters, we can increase the maximum cell density compared to conventional static culture methods. One of the biggest hurdles that remains is an efficient harvesting method that can be scaled up to clinical applications. Current cell detachment protocols use enzymatic based solutions to remove the cells from the surface of the microcarriers. These methods work well in removing the cells, however, they are very labour intensive as they require many washing steps and taking the reactors offline. Therefore, we looked into an alternative method for the detachment of SKP-SCs from microcarriers that will allow for an inline detachment process. This new method is based on previous research done in our lab using high pH solutions to dissociate aggregates. First we investigated the detachment efficiency in static. Cells were cultured in 6-well plates until confluency and then harvested with solutions ranging from pH 8-9.5. With a pH of 9 and an incubation time of 30 minutes, we were able to recover 75% of cells when compared to traditional enzymatic harvesting. Following this we performed a qualitative analysis on the detachment of the SKP-SCs from the microcarriers to determine if this method has potential. Small 3mL samples were taken and solutions with pHs 8.5, 9, and 9.5 were added and incubated for 30 minutes and agitated every 5 minutes. We found that the cells detached with a high efficiency after 30 minutes with a pH of only 8.5. This was then quantified while maintaining a viability of above 90%. Following this we tested this method in harvesting full 125mL bioreactors. We evaluated different pH, agitation rates, and incubation times. We also assessed the ability of the cells to reattach to microcarriers and continue to expand over several serial passages to ensure there were no negative effects on the cells. Lastly we looked at using this method in our controlled bioreactors to increase the pH without the addition of anything else. Based on our results, increasing the pH of the culture medium can detach the SKP-SCs from microcarriers at a pH as low as 8.5 which allows for minimal cell damage while still detaching cells. We also noted that when the pH gets too high (\u3e9.5), the microcarriers begin to clump together causing large aggregates of microcarriers which could lead to clogging during the filtration steps. With increasing agitation, higher recovery efficiencies can be achieved indicating that this method of cell detachment has potential for large volume processes

Scaled-up expansion of equine cord blood mesenchymal stem cells (MSCs) from stirred suspension bioreactors to 100mL computer controlled stirred suspension bioreactors using computational fluid dynamic modeling

Musculoskeletal injuries are the leading cause of lameness and loss of performance in horses and conventional treatments are often associated with high rates of re-injury. Mesenchymal Stem Cells (MSCs) have shown promise for the treatment of such injuries in horses. Currently, the majority of studies are focused on the use of either bone-marrow derived or adipose-derived MSCs. However, equine cord-blood derived MSCs (eCB-MSCs) also provide a promising alternative, due to their high proliferation potential, ability to differentiate towards the chondrogenic lineage, and comparable immune-modulatory properties. Static adherent culture of eCB-MSCs has limited potential to produce sufficient cell numbers for large-scale research studies and possible commercial distribution. Expansion of cells in stirred suspension bioreactors using microcarriers as a scaffold has the potential to generate a large number of cells, using a significantly smaller space, under highly controlled conditions, with reduced time, labour, and monetary requirements. A robust protocol is required for the expansion of eCB-MSCs for use in large research studies and commercial applications. Initially, the hydrodynamic environment in the 10mL and the 100mL bioreactors was modeled using COMSOL Multiphysics software. The volume distributions of shear stress and energy dissipation rate in the bioreactors were calculated and used to determine the operating conditions that would create similar conditions within both scales of bioreactors. Next, eCB-MSCs were expanded in 10mL stirred suspension bioreactors and run at 60rpm and 80rpm with two different impeller geometries: paddles and rounded edges. The bioreactors were loaded at 4500 cells/cm2, and 2g/L microcarriers. The cells at different operating conditions in the 10mL bioreactors achieved varying population doubling times ranging from 0.8d to 1.1d with an average of 0.9d and initial cell attachment ranging from 5000 cells/cm2 to 7700 cells/cm2. The different speeds and geometries produced varying results with maximum attached cell densities from 35,000 to 50,000 cells/cm2 in the bioreactors, compared to maximum cell densities of 44,000 cells/cm2 achieved instatic growth. The expansion of eCB-MSCs was then scaled up in 100mL stirred suspension bioreactors with no direct pH or dissolved oxygen control, using 4500 cells/cm2 and 2g/L microcarriers, with a speed of 40rpm. At this larger scale, the initial cell attachment was 6900 cells/cm2 compared to 6300 cells/cm2 for the 10mL bioreactor. With respect to initial cell attachment, the 100mL bioreactor at 40rpm was most similar to the condition of 80rpm with round edge impeller geometry. The highest attached cell density in the 100 mL vessel was 70,000 cells/cm2. The 100mL uncontrolled bioreactor at 40rpm achieved the most similar results to the 10mL bioreactor run at 60rpm with paddled geometry, with respect to population doubling time with a doubling time of 0.93d for the 10mL bioreactor compared to 0.92d for the 100mL bioreactor. Finally, the eCB-MSCs were expanded in 100mL stirred suspension bioreactors at 4500 cells/cm2, 2g/L and 40rpm with pH and oxygen controlled at 7.4 and 21% DO, respectively, using the DASGIP bioreactor control system. This series of experiments revealed that eCB-MSCs can be expanded in stirred suspension bioreactors

Protocol development to overcome bioprocess bottlenecks in the large-scale expansion of high quality hIPSC aggregates in vertical-wheel bioreactors

Human-induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and trilineage differentiation. hiPSCs are cultured as adherent colonies at small scale, which is sufficient to generate cells for experimental purposes but impractical to achieve large quantities for clinical applications. Bioreactor-based processes are the method of choice for efficient expansion and differentiation of cells. Current protocols for the expansion of hiPSCs, however, utilize horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell-culture starting populations and achieve only moderate cell fold increases within the bioreactor. We have recently demonstrated that the vertical-wheel bioreactor produces a unique fluid flow pattern that results in a homogeneous distribution of hydrodynamic forces, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. Please click Additional Files below to see the full abstract

Computational fluid dynamic characterization of vertical-wheel bioreactors used for effective scale-up of human induced pluripotent stem cell aggregate culture

Innovations in engineering and bioprocess development have accelerated the transition of induced pluripotent stem cell (iPSC) cultivation and use from the bench-top to large-scale clinical manufacturing. Owing to their potency, proliferation capabilities, and ability to overcome the challenges associated with traditional sources of pluripotent stem cells (PSCs), iPSCs have generated significant interest in the field of regenerative medicine for more than a decade. However, traditional bench scale methods to expand iPSCs, including petri dishes and T-flasks, are insufficient to achieve clinically relevant numbers. For iPSC treatments, cell dosages will range from 109 – 1012 cells per patient depending on the therapeutic target. To achieve the required number of cells in an effective and scalable manner, bioreactors will need to be used. Induced pluripotent stem cells (iPSCs) have proven to be extremely sensitive to the bioreactor hydrodynamic environment, making the use of suspension bioreactors to produce quality-assured cells at clinical and commercial scales very challenging. The PBS vertical-wheel (VW) bioreactor combines radial and axial flow components to produce uniform hydrodynamic force distributions, making it a promising platform to overcome the scale-up challenges associated with iPSCs. Please click Additional Files below to see the full abstract

Computational fluid dynamic modeling of 100ml and scaled-down 10ml stirred suspension bioreactors enables prediction of embryonic stem cell characteristics

There is a growing necessity for cell cultivation using bioreactors to translate laboratory based culture protocols into reproducible, scalable, and robust bioprocesses. Stirred suspension bioreactors offer several advantages over planar static cultures, including: reduced labour and operating costs, reduced space requirements, greater cellular homogeneity, and increased cell density per volume [1]. An important consideration when using stirred suspension bioreactors is mechanical stimulation. Fluid shear at the fluid-cell interface triggers cellular responses through mechanotransduction and can modulate stem cell proliferation and differentiation. However, if the shear stress caused by the impeller exceeds the tolerance limit of the cells, it causes cell damage and death, resulting in a lower quality and yield of cells. The shear rate distribution depends on bioreactor geometry, impeller agitation rate, cell density, and cell media viscosity [2]. Current scale-up protocols to predict agitation rates rely on maximum values of hydrodynamic variables, which occur only at the impeller tip. The volume averaged shear stress and maximum shear stress differ greatly, and cells dispersed within the liquid experience different local and global forces. This makes it difficult to predict how cells will respond to changes in bioreactor geometries and sizes. Profiling distributed and average forces in the bioreactor is critical to ensure quality and yield in cell manufacturing. Hydrodynamics, specifically velocities, shear rates, and energy dissipation rates, can be studied using computational fluid dynamic (CFD) modeling. Please click Additional Files below to see the full abstract

Cell Therapy in Veterinary Medicine as a Proof-of-Concept for Human Therapies: Perspectives From the North American Veterinary Regenerative Medicine Association

In the past decade, the potential to translate scientific discoveries in the area of regenerative therapeutics in veterinary species to novel, effective human therapies has gained interest from the scientific and public domains. Translational research using a One Health approach provides a fundamental link between basic biomedical research and medical clinical practice, with the goal of developing strategies for curing or preventing disease and ameliorating pain and suffering in companion animals and humans alike. Veterinary clinical trials in client-owned companion animals affected with naturally occurring, spontaneous disease can inform human clinical trials and significantly improve their outcomes. Innovative cell therapies are an area of rapid development that can benefit from non-traditional and clinically relevant animal models of disease. This manuscript outlines cell types and therapeutic applications that are currently being investigated in companion animals that are affected by naturally occurring diseases. We further discuss how such investigations impact translational efforts into the human medical field, including a critical evaluation of their benefits and shortcomings. Here, leaders in the field of veterinary regenerative medicine argue that experience gained through the use of cell therapies in companion animals with naturally occurring diseases represent a unique and under-utilized resource that could serve as a critical bridge between laboratory/preclinical models and successful human clinical trials through a One-Health approach