Isolation of Porcine Testis Cells from Fresh and Preserved Tissues.
Bibliographic record
Abstract
Gonocytes are the first identifiable germ cells in the testis and progenitors of spermatogonial stem cells. The objective of this study was to maximize gonocyte isolation yield to facilitate the in vitro study and manipulation of these male germline stem cells. Fresh testis tissues from 3-day-old piglets were subjected to mechanical dissociation (Protocols 1: Teasing, 2: Mincing, 3: Sieving, or 4: Grinding), enzymatic digestion (Protocol 5: Collagenase IV (Col)-Dnase1, 6: Col-Dnase1+Col-Dnase1, 7: Col-Dnase1+ Trypsin-EDTA, or 8: Col-Dnase1-Hyaluronidase+Trypsin-EDTA) and enzymatic digestion under cold temperature or pressurized conditions (Protocols 9: Col-Dnase1 for 5-hrs at 4°C followed by 37°C digestion, 10: Col-Hyaluronidase-Dnase1 for 5-hrs at 4°C + Trypsin-EDTA, 11: Pressurized-Col-Dnase1 for 5-hrs at 4°C + 37°C digestion, or 12: Pressurized-Col-Hyaluronidase-Dnase1 for 5-hrs at 4°C +Trypsin-EDTA) (n = 7 replicates each). Additionally, intact testes and testis tissue fragments (30 mg and 100 mg) were maintained at 4°C with PBS, DMEM, Liebovitzs L-15, L-15 with FBS (10%, 20% or 50%), Ham-F10, M199 or HypoThermosol-FRS for 6 days. On preservation Day 0, Day 3 and Day 6, testis tissues were digested (n = 6), and fixed for semi-quantitative morphological analyses (n = 4). Enzymatic methods resulted in the recovery of significantly more testis cells with significantly higher viability than mechanical dissociation methods, while Protocol 10 resulted in the highest cell yield (mean ± SEM, 27.14 ± 0.83×106 per 100-mg testis tissue) and viability (95.91% ± 2.56), Protocol 4 had the lowest viability (36.17% ± 2.29) and Protocol 3 had the lowest cell yield (3.25 ± 0.84×106). Of the enzymatic digestion protocols, Protocol 8 yielded the highest total live cell number (22.72 ± 2.44×106) and its efficiency was significantly increased by cold enzyme treatment (26.02 ± 1.27×106). Compared to fresh tissue dissociation, total live cell number collected on Day 3 and Day 6 dropped significantly, with HTS-FRS (50.43% ± 16.65, 26.19% ± 17.94) and 20% FBS L-15 (45.94% ± 17.52, 21.43% ± 11.19) consistently resulting in the highest numbers. These two media also resulted in the best preservation of morphology. In conclusion, digestion of testis tissue with Col-Dnase1-Hyaluronidase followed by Trypsin-EDTA appears superior to other methods and could be enhanced by adding the enzymes during the cold temperature storage, prior to digestion at 37°C. Cooling of testis tissue may decrease the total live cell recovery by approximately 50% by Day 3 and 75% by Day 6. (poster)
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".