Molecular Design of Synthetic Biodegradable Polymersas Cell Scaffold Materials

Poly(lactic acid) and its copolymers are regarded as the most useful biomaterials. The good biocompatibility, biodegradability and mechanical properties of them make the synthetic biodegradable polymers have primary application to tissue engineering. The advantages and disadvantages of the synthetic biodegradable polymers as cell scaffold materials are evaluated. This article reviews the modification of polylactide-family aliphatic polymers to improve the cell affinity when the polymers are used as cell scaffolds. We have developed four main approaches: to modify polyester cell scaffolds in combination of plasma treating and collagen coating; to introduce hydrophilic segments into aliphatic polyester backbones; to introduce pendant functional groups into polyester chains; to modify polyester with dextran. The results of the cell cultures prove that the approaches mentioned above have improved the cell affinity of the polyesters and have modulated cell function such as adhesion, proliferation and migration.

Mild hypothermia combined with a scaffold of Ng Rsilenced neural stem cells/Schwann cells to treat spinal cord injury

Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differentiation, a cell-scaffold complex seeded with Nogo receptor （NgR）-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly（D,L-lactide-co-glycolic acid） （PLGA） scaffold seeded with NgR-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the NgR-silencing cells ＋ PLGA group, hindlimb motor function and nerve electrophysiological function were dearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve fibers were increased in the group treated with the NgR-silenced cell scaffold ＋ mild hypothermia at 34℃ for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviving cells and nerve fibers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with NgR gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury.

Three-dimensional bioprinting collagen/silk fibroin scaffold combined with neural stem cells promotes nerve regeneration after spinal cord injury

Many studies have shown that bio-scaffolds have important value for promoting axonal regeneration of injured spinal cord.Indeed,cell transplantation and bio-scaffold implantation are considered to be effective methods for neural regeneration.This study was designed to fabricate a type of three-dimensional collagen/silk fibroin scaffold (3D-CF) with cavities that simulate the anatomy of normal spinal cord.This scaffold allows cell growth in vitro and in vivo.To observe the effects of combined transplantation of neural stem cells (NSCs) and 3D-CF on the repair of spinal cord injury.Forty Sprague-Dawley rats were divided into four groups: sham (only laminectomy was performed),spinal cord injury (transection injury of T10 spinal cord without any transplantation),3D-CF (3D scaffold was transplanted into the local injured cavity),and 3D-CF + NSCs (3D scaffold co-cultured with NSCs was transplanted into the local injured cavity.Neuroelectrophysiology,imaging,hematoxylin-eosin staining,argentaffin staining,immunofluorescence staining,and western blot assay were performed.Apart from the sham group,neurological scores were significantly higher in the 3D-CF + NSCs group compared with other groups.Moreover,latency of the 3D-CF + NSCs group was significantly reduced,while the amplitude was significantly increased in motor evoked potential tests.The results of magnetic resonance imaging and diffusion tensor imaging showed that both spinal cord continuity and the filling of injury cavity were the best in the 3D-CF + NSCs group.Moreover,regenerative axons were abundant and glial scarring was reduced in the 3D-CF + NSCs group compared with other groups.These results confirm that implantation of 3D-CF combined with NSCs can promote the repair of injured spinal cord.This study was approved by the Institutional Animal Care and Use Committee of People’s Armed Police Force Medical Center in 2017 (approval No.2017-0007.2).

Selected suitable seed cell, scaffold and growth factor could maximize the repair effect using tissue engineering method in spinal cord injury

Spinal cord injury usually leads to permanent disability, which could cause a huge financial problem to the patient. Up to now there is no effective method to treat this disease. The key of the treatment is to enable the damage zone axonal regeneration and luckily it could go through the damage zone; last a connection can be established with the target neurons. This study attempts to combine stem cell, material science and genetic modification technology together, by preparing two genes modified adipose-derived stem cells and inducing them into neuron direction; then by compositing them on the silk fibroin/chitosan scaffold and implanting them into the spinal cord injury model, seed cells can have features of neuron cells. At the same time, it could stably express the brain-derived neurotrophic factor and neurotrophin-3, both of which could produce synergistic effects, which have a positive effect on the recovery of spinal cord. The spinal cord scaffold bridges the broken end of the spinal cord and isolates with the surrounding environment, which could avoid a scar effect on the nerve regeneration and provide three-dimensional space for the seed cell growth, and at last we hope to provide a new treatment for spinal cord injury with the tissue engineering technique.

Collagen-chitosan scaffold impregnated with bone marrow mesenchymal stem cells for treatment of traumatic brain injury

Combinations of biomaterials and cells can effectively target delivery of cells or other therapeutic factors to the brain to rebuild damaged nerve pathways after brain injury.Porous collagen-chitosan scaffolds were prepared by a freeze-drying method based on brain tissue engineering.The scaffolds were impregnated with rat bone marrow mesenchymal stem cells.A traumatic brain injury rat model was established using the 300 g weight free fall impact method.Bone marrow mesenchymal stem cells/collagen-chitosan scaffolds were implanted into the injured brain.Modified neurological severity scores were used to assess the recovery of neurological function.The Morris water maze was employed to determine spatial learning and memory abilities.Hematoxylin-eosin staining was performed to measure pathological changes in brain tissue.Immunohistochemistry was performed for vascular endothelial growth factor and for 5-bromo-2-deoxyuridine(BrdU)/neuron specific enolase and BrdU/glial fibrillary acidic protein.Our results demonstrated that the transplantation of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds to traumatic brain injury rats remarkably reduced modified neurological severity scores,shortened the average latency of the Morris water maze,increased the number of platform crossings,diminished the degeneration of damaged brain tissue,and increased the positive reaction of vascular endothelial growth factor in the transplantation and surrounding areas.At 14 days after transplantation,increased BrdU/glial fibrillary acidic protein expression and decreased BrdU/neuron specific enolase expression were observed in bone marrow mesenchymal stem cells in the injured area.The therapeutic effect of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds was superior to stereotactic injection of bone marrow mesenchymal stem cells alone.To test the biocompatibility and immunogenicity of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds,immunosuppressive cyclosporine was intravenously injected 12 hours before transplantation and 1-5 days after transplantation.The above indicators were similar to those of rats treated with bone marrow mesenchymal stem cells and collagen-chitosan scaffolds only.These findings indicate that transplantation of bone marrow mesenchymal stem cells in a collagen-chitosan scaffold can promote the recovery of neuropathological injury in rats with traumatic brain injury.This approach has the potential to be developed as a treatment for traumatic brain injury in humans.All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University,China(approval No.AEEI-2015-035)in December 2015.

Morphological properties and proliferation analysis of olfactory ensheathing cells seeded onto three-dimensional collagen-heparan sulfate biological scaffolds

This study aimed to examine the differences in the morphological properties and proliferation of olfactory ensheathing cells in three-dimensional culture on collagen-heparan sulfate biological scaffolds and in two-dimensional culture on common flat culture plates. The proliferation rate of olfactory ensheathing cells in three-dimensional culture was higher than that in two-dimensional culture, as detected by an M-I-r assay. In addition, more than half of the olfactory ensheathing cells subcultured using the trypsinization method in three-dimensional culture displayed a spindly Schwann cell-like morphology with extremely long processes, while they showed a flat astrocyte-like morphology in two-dimensional culture. Moreover, spindle-shaped olfactory ensheathing cells tended to adopt an elongated bipolar morphology under both culture conditions. Experimental findings indicate that the morphological properties and proliferation of olfactory ensheathing cells in three-dimensional culture on collagen-heparan sulfate biological scaffolds are better than those in two-dimensional culture.

Immobilization of Decellularized Valve Scaffolds with Arg-Gly-Asp-containing Peptide to Promote Myofibroblast Adhesion

The cell adhesive properties of decellularized valve scaffolds were promoted by immobilization of valve scaffold with arginine-glycine-aspartic acid （RGD）-containing peptides. Porcine aortic valves were decellularized with trypsin/EDTA, and detergent Triton X-100. With the help of a coupling reagent Sulfo-LC-SPDP, the valve scaffolds were immobilized with glycine-arginine-glycine-aspartic acid-serine-proline-cysteine （GRGDSPC） peptide. X-ray photoelectron spectroscopy （XPS） was used for surface structure analysis. Myofibroblasts harvested from rats were seeded onto the valve scaffolds. Cell count by using microscopy and modified MTT assay were performed to assess cell adhesion. Based on the spectra of XPS, the conjugation of GRGDSPC peptide with decellularized valve scaffolds was confirmed. Both cell count and MTT assay showed that myofibroblasts were much easier to adhere to the modified valve scaffolds, which was also confirmed histologically. Our findings suggest that it is feasible to immobilize RGD-containing peptides onto decellularized valve scaffolds. And the technique can effectively promote cell adhesion, which is beneficial for in vitro tissue engineering of heart valves.

Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

The keratoprosthesis(KPro;artificial cornea)is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision.The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty.The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea.Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells,modification of keratoprosthesis to support cornea cells remains elusive.Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials,but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase.The necrosis of stroma and spontaneous extrusion of the device,allow for maintenance of a precorneal tear layer,and play the role of ensuring a good optical surface and resisting bacterial infection.As a result,improvement in corneal cells has been the main aim of several recent investigations;some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells.The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.

Human amniotic epithelial cells combined with silk fibroin scaffold in the repair of spinal cord injury

Treatment and functional reconstruction after central nervous system injury is a major medical and social challenge. An increasing number of researchers are attempting to use neural stem cells combined with artificial scaffold materials, such as fibroin, for nerve repair. However, such approaches are challenged by ethical and practical issues. Amniotic tissue, a clinical waste product, is abundant, and amniotic epithe- lial cells are pluripotent, have low immunogenicity, and are not the subject of ethical debate. We hypothesized that amniotic epithelial cells combined with silk fibroin scaffolds would be conducive to the repair of spinal cord injury. To test this, we isolated and cultured amniotic epithelial cells, and constructed complexes of these cells and silk fibroin scaffolds. Implantation of the cell-scaffold complex into a rat model of spinal cord injury resulted in a smaller glial scar in the damaged cord tissue than in model rats that received a blank scaffold, or amniotic epithelial cells alone. In addition to a milder local immunological reaction, the rats showed less inflammatory cell infiltration at the trans- plant site, milder host-versus-graft reaction, and a marked improvement in motor function. These findings confirm that the transplantation of amniotic epithelial ceils combined with silk fibroin scaffold can promote the repair of spinal cord injury. Silk fibroin scaffold can provide a good nerve regeneration microenvironment for amniotic epithelial cells.

Liver regeneration using decellularized splenic scaffold: a novel approach in tissue engineering

BACKGROUND： The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration. METHODS： After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×106 bone marrow mesenchymal stem cells（BMSCs） was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.RESULTS： After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The poros-ity of scaffold, aperture of 45.36±4.87 μm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix. CONCLUSION： Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

Growth and differentiation of neural stem cells in a three-dimensional collagen gel scaffold

Collagen protein is an ideal scaffold material for the transplantation of neural stem cells. In this study rat neural stern cells were seeded into a three-dimensional collagen gel scaffold, with suspension cultured neural stem cells being used as a control group. Neural stem cells, which were cultured in medium containing epidermal growth factor and basic fibroblast growth factor, actively expanded and formed neurospheres in both culture groups. In serum-free medium conditions, the processes extended from neurospheres in the collagen gel group were much longer than those in the suspension culture group. Immunofluorescence staining showed that neurespheres cultured in collagen gels were stained positive for nestin and differentiated cells were stained positive for the neuronal marker βIII-tubulin, the astrocytic marker glial fibrillary acidic protein and the oligodendrocytic marker 2＇,3＇-cyclic nucleotide 3＇-phosphodiesterase. Compared with neurospheres cultured in suspension, the differentiation potential of neural stem cells cultured in collagen gels increased, with the formation of neurons at an early stage. Our results show that the three-dimensional collagen gel culture system is superior to suspension culture in the proliferation, differentiation and process outgrowth of neural stem cells.

Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells （PSCs） into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group （P〈0.05）. Quantitative real-time PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group （P〈0.01）. ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group＇s PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated （P〈0.01）. The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.

Aligned Fibrous Scaffold Induced Aligned Growth of Corneal Stroma Cells in vitro Culture

To investigate the contribution of fibre arrangement to guiding the aligned growth of corneal stroma cells,aligned and randomly oriented fibrous scaffolds of gelatin and poly-L-lactic acid（PLLA） were fabricated by electrospinning.A comparative study of two different systems with corneal stroma cells on randomly organized and aligned fibres were conducted.The efficiency of the scaffolds for inducing the aligned growth of cells was assessed by morphological observation and 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl-tetrazolium bromide（MTT） assay.Results show that the cells cultured on both randomly oriented and aligned scaffolds maintained normal morphology and well spreading as well as long term proliferation.Importantly,corneal stroma cells grew high orderly on the aligned scaffold,while the cells grew disordered on the randomly oriented scaffold.Moreover,the cells exhibited higher viability in aligned scaffold than that in randomly oriented scaffold.These results indcate that electrospinng to prepare aligned fibrous scaffolds has provided an effective approach to the aligned growth of corneal stroma cells in vitro.Our findings that fiber arrangement plays a crucial role in guiding the aligned growth of cells may be helpful to the development of better biomaterials for tissue engineered cornea.

Extracellular matrices, artificial neural scaffolds and the promise of neural regeneration

Over last 20 years, extracellular matrices have been shown to be useful in promoting tissue regeneration. Recently, they have been used and have had success in achieving neurogenesis. Recent developments in extracellular matrix design have allowed their successful in vivo incorporation to engender an environment favorable for neural regeneration in animal models. Promising treatments under investigation include manipulation of the intrinsic extracellular matrix and incorporation of engineered naometer-sized scaffolds through which inhibition of molecules serving as barriers to neuroregeneration and delivery of neurotrophic factors and/or cells for successful tissue regeneration can be achieved. Further understanding of the changes incurred within the extracellular matrix following central nervous system injury will undoubtedly help design a clinically efficacious extracellular matrix scaffold that can mitigate or reverse neural degeneration in the clinical setting.

A Novel <i>in Vitro</i>Three-Dimensional Macroporous Scaffolds from Bacterial Cellulose for Culture of Breast Cancer Cells

In this work, patterned macropores with a diameter larger than 100 μm were introduced to pristine three-dimensional (3D) nanofibrous bacterial cellulose (BC) scaffolds by using the infrared laser micromachining technique in an attempt to create an in vitro model for the culture of breast cancer cells. The morphology, pore structure, and mechanical performance of the obtained patterned macroporous BC (PM-BC) scaffolds were characterized by scanning electron microscopy (SEM), mercury intrusion porosimeter, and mechanical testing. A human breast cancer cell (MDA-MB-231) line was cultured onto the PM-BC scaffolds to investigate the role of macropores in the control of cancer cell behavior. MTT assay, SEM, and hematoxylin and eosin (H&E) staining were employed to determine cell adhesion, growth, proliferation, and infiltration. The PM-BC scaffolds were found to be able to promote cellular adhesion and proliferation on the scaffolds, and further to allow for cell infiltration into the PM-BC scaffolds. The results demonstrated that BC scaffolds with laser-patterned macropores were promising for the in vitro 3D culture of breast cancer cells.

A novel porcine acellular dermal matrix scaffold used in periodontal regeneration

Regeneration of periodontal tissue is the most promising method for restoring periodontal structures.To find a suitable bioactive three- dimensional scaffold promoting cell proliferation and differentiation is critical in periodontal tissue engineering.The objective of this study was to evaluate the biocompatibility of a novel porcine acellular dermal matrix as periodontal tissue scaffolds both in vitro and in vivo.The scaffolds in this study were purified porcine acellular dermal matrix(PADM) and hydroxyapatite-treated PADM(HA-PADM). The biodegradation patterns of the scaffolds were evaluated in vitro.The biocompatibility of the scaffolds in vivo was assessed by implanting them into the sacrospinal muscle of 20 New Zealand white rabbits.The hPDL cells were cultured with PADM or HA-PADM scaffolds for 3,7,14,21 and 28 days.Cell viability assay,scanning electron microscopy(SEM),hematoxylin and eosin(H&E) staining, immunohistochemistry and confocal microscopy were used to evaluate the biocompatibility of the scaffolds.In vitro,both PADM and HA-PADM scaffolds displayed appropriate biodegradation pattern,and also,demonstrated favorable tissue compatibility without tissue necrosis,fibrosis and other abnormal response.The absorbance readings of the WST-1 assay were increased with the time course, suggesting the cell proliferation in the scaffolds.The hPDL cells attaching,spreading and morphology on the surface of the scaffold were visualized by SEM,H&E staining,immnuohjstochemistry and confocal microscopy,demonstrated that hPDL cells were able to grow into the HA-PADM scaffolds and the amount of cells were growing up in the course of time.This study proved that HA-PADM scaffold had good biocompatibility in animals in vivo and appropriate biodegrading characteristics in vitro.The hPDL cells were able to proliferate and migrate into the scaffold.These observations may suggest that HA-PADM scaffold is a potential cell carrier for periodontal tissue regeneration.

Dextran coating on and among fibers of polymer sponge scaffold for osteogenesis by bone marrow cells in vivo

Although hydroxyapatite is commonly used as a scaffold for bone regeneration, sponges may be suitable because of the adaptability to the defect. To use as a scaffold, the fiber of sponge would be coated with any adhesive to storage stem cells in the sponges. Fiber in the structure of commercially available sponges was coated by immersion in dextran solution and air dried. After seeding of rat bone marrow cells (rBMCs), the sponges were implanted subcutis of rats for estimate osteogenesis in vivo. The level of osteocalcin was 25.28 &#177;5.71 ng/scaffold and that of Ca was 129.20 &#177;19.69 μg/scaffold. These values were significantly high- er than those in sponges without dextran coating (p 【0.01). It was thought that rBMCs could be stored on the shelf by dextran deposition in the fiber of the sponge. In vivo examination, dextran induced osteogenesis by rBMCs in many spaces in the inner structure of the sponge.

Immobilization of RGD Peptidcs onto Decellularized Valve Scaffolds to Promote Cell Adhesion

Porcine aortic valves were decellularized with trypsinase/EDTA and Triton-100. With the help of a coupling reagent Sulfo-LC-SPDP, the biological valve scaffolds were immobilized with one of RGD （arginine-glycine-aspartic acid） containing peptides, called GRGDSPC peptide. Myofibroblasts harvested from rats were seeded onto them. Based on the spectra of X-ray photoelectron spectroscopy, we could find conjugation of GRGDSPC peptide and the scaffolds. Cell count by both microscopy and MTT assay showed that myofibroblasts were easier to adhere to the modified scaffolds. It is proved that it is feasible to immobilize RGD peptides onto decellularized valve scaffolds, and effective to promote cell adhesion, which is beneficial for constructing tissue engineering heart valves in vitro.

Spatial perturbation with synthetic protein scaffold reveals robustness of asymmetric cell division

Asymmetric cell division is an important mechanism for creating diversity in a cellular population. Stem cells commonly perform asymmetric division to generate both a daughter stem cell for self-renewal and a more differentiated daughter cell to populate the tissue. During asymmetric cell division, protein cell fate determinants asymmetrically localize to the opposite poles of a dividing cell to cause distinct cell fate. However, it remains unclear whether cell fate determination is robust to fluctuations and noise during this spatial allocation process. To answer this question, we engineered Caulobacter, a bacterial model for asymmetric division, to express synthetic scaffolds with modular protein interaction domains. These scaffolds perturbed the spatial distribution of the PleC-DivJ- DivK phospho-signaling network without changing their endogenous expression levels. Surprisingly, enforcing symmetrical distribution of these cell fate de terminants did not result in symmetric daughter fate or any morphological defects. Further computational analysis suggested that PleC and DivJ form a robust phospho-switch that can tolerate high amount of spatial variation. This insight may shed light on the presence of similar phospho-switches in stem cell asymmetric division regulation. Overall, our study demonstrates that synthetic protein scaffolds can provide a useful tool to probe biological systems for better understanding of their operating principles.