A Cell’s Viscoelasticity Measurement Method Based on the Spheroidization Process of Non-Spherical Shaped Cell

The mechanical properties of biological cells, especially the elastic modulus and viscosity of cells, have been identified to reflect cell viability and cell states. The existing measuring techniques need additional equipment or operation condition. This paper presents a cell’s viscoelasticity measurement method based on the spheroidization process of non-spherical shaped cell. The viscoelasticity of porcine fetal fibroblast was measured. Firstly, we introduced the process of recording the spheroidization process of porcine fetal fibroblast. Secondly, we built the viscoelastic model for simulating a cell’s spheroidization process. Then, we simulated the spheroidization process of porcine fetal fibroblast and got the simulated spheroidization process. By identifying the parameters in the viscoelastic model, we got the elasticity (500 Pa) and viscosity (10 Pa·s) of porcine fetal fibroblast. The results showed that the magnitude of the elasticity and viscosity were in agreement with those measured by traditional method. To verify the accuracy of the proposed method, we imitated the spheroidization process with silicone oil, a kind of viscous and uniform liquid with determined viscosity. We did the silicone oil’s spheroidization experiment and simulated this process. The simulation results also fitted the experimental results well.

Graphene Oxide–Silver Nanoparticle Nanocomposites Induce Oxidative Stress and Aberrant Methylation in Caprine Fetal Fibroblast Cells

Graphene oxide–silver nanoparticle (GO-AgNPs) nanocomposites have drawn much attention for their potential in biomedical uses. However, the potential toxicity of GO-AgNPs in animals and humans remains unknown, particularly in the developing fetus. Here, we reported the GO-AgNP-mediated cytotoxicity and epigenetic alteration status in caprine fetal fibroblast cells (CFFCs). In brief, the proliferation and apoptosis rate of GO-AgNP-treated CFFCs (4 and 8 µg/mL of GO-AgNPs) were measured using the cell-counting kit (CCK-8) assay and the annexin V/propidium iodide (PI) assay, respectively. In addition, the oxidative stress induced by GO-AgNPs and detailed mechanisms were studied by evaluating the generation of reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), and caspase-3 and abnormal methylation. The expression of pro- and anti-apoptotic genes and DNA methyltransferases was measured using reverse transcription followed by RT-qPCR. Our data indicated that GO-AgNPs cause cytotoxicity in a dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, production of ROS, increasing leakage of LDH and level of MDA, increasing expression of pro-apoptotic genes, and decreasing expression of anti-apoptotic genes. GO-AgNPs incited DNA hypomethylation and the decreased expression of DNMT3A. Taken together, this study showed that GO-AgNPs increase the generation of ROS and cause apoptosis and DNA hypomethylation in CFFCs. Therefore, the potential applications of GO-AgNPs in biomedicine should be re-evaluated.

About the effectiveness of cell technologies in extensive soft tissue defects plasty

Fetal fibroblast culture transplantation results were evaluated in the treatment of 18 burn victims. Comparison group consisted of 18 burn patients received medical care without cellular technologies utilization. The main comparison parameters in the study groups: the timing of the first stage of autodermoplasty; the number of autodermoplasties during the treatment; hospitalization duration; the newly formed epidermis area estimation. Fetal fibroblast culture transplantation in burn patients with extensive skin defects was performed on average 14,883,56 days after the injury. The timing of the first stage of autodermoplasty did not differ in the main and control groups, not exceeding an average of 19,122,01 days (p=0,48). An average of 2,710,67 surgeries using cell technologies performed in patients of the study group. The use of fetal fibroblasts culture in patients with extensive skin defects reduces the need for autodermoplasty by 1,6 times due to the granulation tissue formation and the epidermal growth beginning 7 days after and complete epidermal formation 14 days after transplantation. Regenerative medicine technologies utilization in patients with extensive skin lesions is possible and appropriate. Due to the fetal fibroblasts culture transplantation a kind of temporary biological coating is formed in the wound. It accelerates the wound healing process phase change from exudation to proliferation and the preparation of skin defects for autodermoplasty, expanding the possibilities of effective patients treatment.

Phenotypic Differences in Adult and Fetal Dermal Fibroblast Responses to Mechanical Tension

ABSTRACTThe regenerative wound healing phenotype observed in the fetal wounds is characterized by a unique hyaluronan rich wound microenvironment. Fetal fibroblasts produce a pericellular matrix which is abundant in high molecular weight HA. In vivo, fetal skin has negligible resting tension and heals regeneratively when small wounds are made. However, critically large fetal wounds heal with increased scar. We hypothesize that higher mechanical tension will differentially alter fibroblast-mediated HA metabolism in adult and fetal fibroblasts, leading to a profibrotic fetal fibroblast phenotype. C57BL/6J fetal (FFB, E14.5) and adult (AFB, 8 wk) dermal fibroblasts were cultured on silicone membranes +/-10% static strain (1, 3, 6, and 12h). Monolayers were analyzed via PCR for HA-synthesis (HAS1-3), HA-remodeling (HYAL1-2, KIAA1199), and HA-receptor (CD44) genes, normalized to static FFB. Total HA was analyzed by gel electrophoresis at 12h. Data is reported as mean+/-SD (n=3), with p-values calculated by two-way ANOVA (post-hoc Tukey’s test). FFB pericellular matrix was reduced after tension conditions, and HA profile shifted from HMW-HA to LMW-HA. Under static conditions, AFB had increased expression of HAS 1 and 2, as well as increased expression of KIA1199, HYAL1 and 2 when compared to FFB. Overall, tension resulted in an increase in HAS1, HAS3, and HYAL1 expression in FFB, and decreased HAS2 but increased HYAL1 for AFB under tension. Staining for cytoskeletal components demonstrates that tension results in organization of F-actin and aSMA in FFB to resemble AFB. Expression of aSMA is increased in AFB with tension, while CD26 expression is increased in FFB with tension. These insights into the intrinsic differences between regenerative FFB and fibrotic AFB may yield targets to attenuate fibrosis.

Graphene oxide-silver nanocomposites induce oxidative stress and apoptosis in caprine fetal fibroblast cells

Background: Graphene oxide (GO) has drawn much attention as excellent platform to which silver nanoparticles (AgNPs) can be anchored for the production of biomedical nanocomposites. Yet, the potential toxicity of reduced graphene oxide- silver nanoparticles (GO-AgNPs) nanocomposites to animal and human is complex to evaluate and remains largely unknown.Results: Our data indicated that GO-AgNPs caused cytotoxicity in dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, over-production of reactive oxygen species (ROS), increased leakage of lactate dehydrogenase (LDH) and level of malondialdehyde (MDA), increased expression of pro-apoptotic genes and decreased expression of anti-apoptotic genes.Conclusions: This study demonstrated that GO-AgNPs potentially induced oxidative stress, which resulted in toxicity and cell apoptosis in caprine fetal fibroblast cell due to an increased generation of ROS. For antibacterial applications of GO-AgNPs nanocomposite in animal, the toxical effect must be further evaluated.

Graphene oxide-silver nanoparticle nanocomposites induces apoptosis in caprine fetal fibroblast cells via induction of oxidative stress

Background: Graphene oxide (GO) has drawn much attention as excellent platform to which silver nanoparticles (AgNPs) can be anchored for the production of biomedical nanocomposites. Yet, the potential toxicity of GO-AgNPs nanocomposites to animal and human is complex to evaluate and remains largely unknown. Results: Our data indicated that GO-AgNPs caused cytotoxicity in dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, production of reactive oxygen species (ROS), cell cycle arrest, increasing leakage of lactate dehydrogenase (LDH) and level of Malondialdehyde (MDA), increasing expression of pro-apoptotic genes and decreasing expression of anti-apoptotic genes. Conclusions: Taken together, our study demonstrated that GO-AgNPs potentially induce oxidative damage to DNA, which result in toxicity and cell apoptosis in caprine fetal fibroblast cell due to an increased generation of ROS. It strongly suggests that applications of GO-AgNPs nanocomposite in animal must be further evaluated.

206 Towards the generation of transchromosomic goats for the production of fully human immunoglobulin

We have previously reported that inactivation of endogenous immunoglobulin (Ig) genes can enhance the production of human polyclonal antibodies (hpAb) in transchromosomic (Tc) cattle containing a human artificial chromosome (HAC) comprising the entire human Ig gene repertoire. Goats offer the advantages of having a shorter gestation period and growing to adult size much faster than larger ungulates. Therefore, based upon our previously established Tc cattle platform, in this study, we aimed to generate immunoglobulin lambda light chain knockout (IGLλ −/− )/HAC Tc goats expressing hpAb using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. First, we designed multiple specific single-guide RNAs (sgRNA) targeting the coding sequences of constant regions of goat IGLλ1, IGLλ2 (both in NC_030824.1), and IGLλ3 (NW_017189530.1) and constructed corresponding gene targeting vectors. Gene targeting efficiency analysis in goat fibroblasts showed that the sgRNA were efficient in directing Cas9 to generate targeted indels in corresponding IGLλ genes with efficiencies ranging between 20 and 30%. We then targeted the IGLλ genes and generated a cloned IGLλ −/− fetus by SCNT (first round of cloning; G1) using single cell-derived IGLλ −/− fibroblast colonies as nuclear donors and established IGLλ −/− fetal fibroblast cell lines. Second, we established IGLλ −/− /HAC fibroblast colonies by transferring the isKcHACΔ, a HAC containing the entire unrearranged human immunoglobulin heavy-chain and kappa-chain sequences, into the IGLλ −/− fetal fibroblast cells via microcell-mediated chromosome transfer (MMCT; Matsushita et al. 2014 PLoS One 9, e90383). The resulting IGLλ −/− /HAC fibroblasts were used as nuclear donors for the production of IGLλ −/− /HAC goats by SCNT (second round of cloning; G2). A total of 311 one-cell stage G2 embryos were generated using 4 IGLλ −/− /HAC fetal fibroblast colonies and surgically transferred into 19 oestrus-synchronized recipients. Seven pregnancies were confirmed (7/19; 36.8%) with ultrasonography at Day 60±3 of gestation, and 1 pregnancy (1/7; 14.3%) developed to term, leading to the birth of a live and healthy kid. Genetic and serological characterisation of this Tc goat produced by the second round of SCNT is in progress.

28 Generation of Immunoglobulin Heavy Constant Mu (IGHM) Knockout Goats Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer

Towards the goals of inactivating endogenous goat immunoglobulin (Ig) genes and producing fully human polyclonal antibodies in transchromosomal goats carrying a human artificial chromosome comprising the entire human Ig gene repertoire, we report here the successful generation of IgM heavy chain knockout (IGHM−/−) goats using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. Two single-guide RNAs (sgRNAs) were designed specific for exon 1 of the goat IGHM constant region (GenBank: EU182621.1). The gene-targeting vectors were constructed by using pX330 plasmid (Plasmid No. 42230, Addgene, Cambridge, MA, USA) and transfected into 2 × 105 goat fetal fibroblast cells. Gene-targeting efficiency of each targeting vector was determined by PCR-restriction fragment length polymorphism (RFLP) assay 3 days post-transfection. Our results showed that one of the sgRNAs, 5′-GAAAGGCGCTTGAGGAATGC-3′, was efficient in directing Cas9 to generate targeted cleavages in exon 1 of IGHM constant region, with a mutation efficiency of 20%. We established single cell-derived fetal fibroblast colonies by limiting dilution of the cells transfected with the targeting vector. Colony screening with the PCR-RFLP assay confirmed that we achieved targeted gene disruption in exon 1 in 11/49 (22.4%) of the colonies (7 colonies with biallelic and 4 with monoallelic gene disruption). Sanger sequencing analysis of genomic DNA isolated from cell colonies with biallelic mutations showed that typical nucleotide deletions and insertions (indels), caused by repairing double-strand DNA breaks during the error-prone non-homologous end joining (NHEJ) process, were generated at the targeting site of exon 1. One of the colonies harboring a 1-nucleotide (nt) deletion was used as nuclear donors for SCNT. A total of 102 1-cell-stage cloned embryos were generated and surgically transferred into 6 synchronized recipients. Three of the recipients (3/6, 50.0%) were confirmed pregnant by ultrasonography on Day 40 to 45 of gestation. Two pregnancies were sacrificed for IGHM−/− fetal fibroblast isolation and one pregnancy was allowed to go to term, which led to the birth of a live and seemingly healthy kid. This goat was reared conventionally and appeared healthy until 5 weeks of age when it was killed because of commensal virus infection. The PCR-RFLP assay and sequence analysis showed that this cloned goat carried a biallelic 1-nt deletion in exon 1 of IGHM, which was identical to the donor colony it was originated from. No lymphoid follicles were observed in lymph nodes and spleen by histology, and immunohistochemistry for B cells (CD20) and T cells (CD3) demonstrated a lack of B cells in lymph nodes and spleen but the presence of T cells, confirming that IGHM has been successfully knocked out.