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Article by Ylleana Goduco, Maria Hudock & Margaretha Morsink
Optimizing Human Kidney Decellularization with SDS
Source Publication:
Efficient decellularization of human fetal kidneys through optimized SDS exposure, Scientific Reports, 2024
Mohamad Hossein Khosropanah et al., Abdol-Mohammad Kajbafzadeh Lab.
Chronic kidney disease affects millions of people worldwide, and despite current treatments, many patients still face the risk of kidney failure, creating an urgent need for new therapies. Decellularization is a promising technique involving the removal of cells from the kidney, which leaves behind a cell-free scaffold that can be recellularized with the transplant recipient's own cells. The scaffold, called the extracellular matrix (ECM) is essential for maintaining the organ's structure and provides a healthy environment for new cells to grow and thrive.. However, there are a few challenges with decellularization, including the resistance of cells to decellularization detergents, the need to preserve ECM integrity, and the necessity of maintaining ECM networks that act as anchors to reseeded cells during recellularization. Decellularization must reach the balance between sufficient disruption to effectively remove cells while minimizing damage to the ECM. This paper seeks to optimize the decellularization process by examining cell resistance to detergents and the retention of the extracellular matrix (ECM). Specifically, it evaluates tissue responses to the decellularization agent sodium dodecyl sulfate (SDS).
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What did these researchers do?
In order to find the optimal conditions for decellularization, the researchers started with a common decellularization agent: SDS. SDS disrupts the lipid bilayer and lipid-lipid hydrophobic interactions, effectively solubilizing cell membranes. This property makes it useful for shortening the perfusion duration required for decellularization. However, higher SDS concentrations can also denature growth factors within the extracellular matrix (ECM), potentially compromising its functionality.
To reach the balance between decellularization and maintaining the ECM, the study tested the effectiveness of varying SDS exposures on decellularization. The researchers worked with six kidneys, dividing them into two groups based on perfusion duration: three kidneys were perfused for 24 hours, and the other three for 48 hours. Within each group, the kidneys were further treated with three different SDS concentrations: 0.1%, 0.3%, and 0.5%. Through imaging and immunohistochemistry (IHC) staining, this study found the optimal SDS exposure to be 0.1% for 24 hours.
Why is this important?
Chronic kidney disease is estimated to impact 37 million people in the US. It begins with patients classified as high-risk, progressing to chronic kidney disease, and in severe cases, advancing to end-stage renal disease (ESRD). The primary treatment for ESRD is renal replacement therapy, which involves transplantation and dialysis (or hemodialysis). However, with the number of patients on the transplant waitlist steadily increasing, there’s a growing need to explore other options in the regenerative medicine and tissue engineering sphere. Kidney decellularization is a promising option that can help bridge the gap between organ demand and availability. Decellularization, followed by recellularization, creates a functional organ that can be transplanted into the patient. This technique works by first removing the native cells from donor kidneys using decellularizing agents perfused through the organ's existing vasculature. Removing native cells reduces the risk of immune rejection, as cellular components are typically the primary triggers of immune responses to transplanted organs. Following decellularization, the kidney scaffold is recellularized with the patient’s cells, which is an approach that could eliminate the need for lifelong immunosuppressive drugs that cause patients to become more susceptible to infections and other illnesses. By recellularizing the kidney scaffold with the patient’s cells, this decellularization-recellularization method could eliminate the need for immunosuppressive drugs.
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How did the researchers do this?
The researchers began by rinsing the kidneys to remove excess blood and tissue. They then cannulated the organ, and flushed phosphate-buffered saline (PBS) to wash out any remaining blood inside the kidney. To ensure decontamination and sterilization, they used SDS decellularization solutions with antibiotics, such as penicillin, streptomycin, and antifungal amphotericin. A peristaltic pump was used to perfuse the kidneys with the respective treatments, varying by perfusion duration (24 hours vs. 48 hours) and SDS concentration (0.1%, 0.3%, and 0.5%).
After decellularization, the SDS was flushed out, and a colorimetric assay was conducted to confirm effective removal of the detergent. To assess the kidney scaffold’s structure post-decellularization, CT-angiography was used to image the blood vessels. To evaluate the effectiveness of decellularization, histopathology was used to compare the cell counts between treated and untreated tissues in order to determine the cell survival rate. Immunofluorescent staining with 4′,6-diamidino-2-phenylindole (DAPI) was used to detect the presence of intact cells remaining in the scaffold, which also provides data for the success of decellularization. Since preserving the ECM is vital, immunohistochemistry (IHC) staining was used to verify if major ECM components (elastin, laminin, collagen I, and collagen IV) remained in the scaffold.
Immunofluorescence showing the 6 different kidneys that were decellularized. The blue staining (DAPI) indicates that cells are remaining. The green staining shows the extracellular matrix
What comes next?
In this study, researchers were able to address one of the critical limitations in developing prosthetic implants by developing a biomimetic engineered bone platform to test the osseointegration potential of various implants. While this is great progress, there are some limitations that need to be addressed. The first is the inclusion of more cell types as the current system only involves iMSCs in a bone scaffold. In the body, there are many other processes that occur when receiving an implant, such as response from immune cells. Thus, they should include other cell types to create a more comprehensive model. Another possible direction is to test other mechanical factors on their system, since physical stimulation like load-bearing and stress can also affect osseointegration.
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