Use cases
2.5×10⁵ cells in 10 μL into proximal tibial marrow models established osteolysis and tumor–bone crosstalk without homing. Contralateral PBS sham. Watch JoVE—not a substitute for surgical training.
Catalog: MC-h133 · Product modeling page · In-vitro spheroid
RUO. IACUC, asepsis, and analgesia per protocol. No anesthetic doses here. Watch the JoVE tibia segment. This is not the LV SOP.
Campbell JoVE: intratibial (same video as LV) · PMID 22972196
Use-case overview
Use this page for local osteolysis/bone-targeted agents; switch to LV for homing. Do not put the 100 μL LV volume into the marrow.
Does the workflow match?
Local osteolysis is this page; homing is LV.
| Use | vs foolproof SOP | Differences vs core SOP |
|---|---|---|
| Post-colonization bone disease | Same core SOP | This page. |
| Experimental bone mets (with homing) | Other route | Left ventricle, 100 μL, not 10 μL. |
In one line
IACUC → prepare cells → tibial drill/inject → imaging/clinical signs → bone pathology.
D−1 / D0 timeline
1. D−1
Feed at 80–90% confluence.
2. D0
2.5×10⁵ / 10 μL → one tibia; PBS contralateral.
3. From D+7
Lameness, weight, X-ray.
Protocol overview
Scenario-specific flowchart (core engraftment skeleton with host/therapy or imaging/readout changes).
- IACUC + analgesia SOP
- Expand MDA-MB-231 (T75 on expansion table)
- Anesthetize; patellar ligament → proximal tibia
- Inject 10 μL into marrow (PBS contralateral)
- X-ray / μCT monitor
- Endpoint histology
Novice pack (literature cases)
Flowchart buttons open the matching table. Full pack below, in use order.
Unpack list (intratibial, plan per mouse)
Marrow-cavity local inoculum—not LV, not tail vein. Watch the tibia segment of the same JoVE. No Matrigel.[1]
| Item | Per mouse / study | Notes |
|---|---|---|
| MDA-MB-231 MC-h133 | Start from 1 vial | STR/mycoplasma-qualified |
| FBS MC100 | 10% complete medium | Regular grade |
| Ice-cold PBS | 10 μL/side | Contralateral often 10 μL PBS sham[1] |
| 28G ½″ needle | 1 per side | Through the patellar ligament into proximal tibia; if clogged, new needle along the tract[1][2] |
| Analgesia / asepsis | Per IACUC | No doses on this page |
Expansion cases: mice vs T75 (tibia 2.5×10⁵)
JoVE 2.5×10⁵ / 10 μL + 20%. Ten mice still ~1 T75. Do not reuse LV 100 μL or orthotopic 2×10⁶ math.[1]
| Mice | T75 flasks |
|---|---|
| 1 | 1 |
| 2 | 1 |
| 3 | 1 |
| 4 | 1 |
| 5 | 1 |
| 6 | 1 |
| 7 | 1 |
| 8 | 1 |
| 9 | 1 |
| 10 | 1 |
Inoculum recipe (small marrow volume)
JoVE: 2.5×10⁵ in 10 μL ice-cold PBS. Contralateral 10 μL PBS.[1]
Surgery checklist (proximal tibial marrow)
Knee ~90°; drill through the growth plate ~2–3 mm via the patellar ligament; a marrow needle will not swing freely. Free swing = muscle.[1][2]
| Check | Pass | If fail |
|---|---|---|
| Approach | Patellar white line, intercondylar area | Unclear: optional 2–3 mm skin nick (Wright)[2] |
| Depth | Give after the growth plate, ~2–3 mm[2] | Swings freely: withdraw and re-enter—do not dump cells into soft tissue |
| Push | Slow 10 μL, little resistance[1] | High resistance: not in marrow or clogged—new needle along the tract[2] |
| Contralateral | Same motion with PBS[1] | Cells in both legs lose the internal sham |
Culture card (MDA-MB-231 / MC-h133)
Inverted microscope (schematic 10× field): confluence is the % of the growth surface covered by cells. If the monolayer is even, that matches the fraction of the field occupied. Split/harvest at the middle panel. Click the schematic to enlarge.
Same harvest as LV: 80–90% confluence, ice-cold PBS, 30 min.[1]
What success looks like
Troubleshooting
Soft-tissue tumor = spill, not a marrow model.
| Sign | Cause | Action |
|---|---|---|
| Peri-knee mass | Intramuscular | Log that side as failed; check for a swinging needle |
| Acute embolus signs | Clumps (less common than LV) | Stop the cohort; inspect the mix |
| Bilateral osteolysis | Cells in the sham leg | Sham is void; re-pilot |
Monitoring log fields
Split left/right tibia. Lameness outranks the image.
| Field | How to log |
|---|---|
| Side | Cells vs PBS |
| Lameness / weight | Stop if endpoint hit |
| X-ray/μCT | Proximal tibia |
Literature case comparison
- 01[Ethics] IACUC; analgesia and asepsis. RUO. Watch the JoVE tibia segment[1]. No anesthetic doses.
Marrow-cavity local inoculum—not LV, not tail vein. Watch the tibia segment of the same JoVE. No Matrigel.[1]
Item Per mouse / study Notes MDA-MB-231 MC-h133 Start from 1 vial STR/mycoplasma-qualified FBS MC100 10% complete medium Regular grade Ice-cold PBS 10 μL/side Contralateral often 10 μL PBS sham[1] 28G ½″ needle 1 per side Through the patellar ligament into proximal tibia; if clogged, new needle along the tract[1][2] Analgesia / asepsis Per IACUC No doses on this page - 02[Cells] 80–90% confluence; 2.5×10⁵ / 10 μL ice-cold PBS; no Matrigel; finish in 30 min[1].
- 03
- 04[Monitor] Lameness, weight, X-ray. Watch paraplegia days 21–28[1].
Split left/right tibia. Lameness outranks the image.
Field How to log Side Cells vs PBS Lameness / weight Stop if endpoint hit X-ray/μCT Proximal tibia Soft-tissue tumor = spill, not a marrow model.
Sign Cause Action Peri-knee mass Intramuscular Log that side as failed; check for a swinging needle Acute embolus signs Clumps (less common than LV) Stop the cohort; inspect the mix Bilateral osteolysis Cells in the sham leg Sham is void; re-pilot - 05[Endpoint] Decalcified HE, TRAP; cells vs PBS side.
- 06[Interpretation] Post-colonization bone disease only—not homing.
Reagents / materials
| Item | Role | Conc. / dose |
|---|---|---|
| MDA-MB-231 cells (MC-h133) | Tibial marrow inoculum | 2.5×10⁵ / 10 μL 冰 PBS[[1]] |
| Anesthetic / analgesic | Surgery & welfare | 按伦理批件与兽医 SOP |
| Test drug / vehicle (optional) | Treatment arm | 按药理方案 |
Readouts
Local osteolysis scores, μCT bone metrics, histologic burden, lameness/weight.
References (PubMed)
- [1]PMID 22972196 — Models of bone metastasis. J Vis Exp (2012)
- [2]PMID 27867497 — Murine models of breast cancer bone metastasis. Bonekey Rep (2016)
Disclaimer: RUO; IACUC required. Optimize by strain and pilot. Red tags mark weak or non-metastatic parental endpoints.