We follow both major lines of processing which have emerged –
1- One advocating stem cell enrichment
2- The other doing expansion with or without differentiating these endogenous stem cells.
Enrichment has the advantage of fresh cells along with cocktail of numerous different types of supportive cells and growth factors neseccary for stem cells to home at lesion and their growth.
Expansion /Culture of stem cells increases the number significantly so as to reach desired number of cells for a therapeutic effect
1- Minimal manipulation by any chemical, physical or biologic agent as these cells are highly sensitive towards process would loosen their properties soon.
2- Fresh i.e. less time delay between extraction –isolation and enrichment
3- Provides cocktail of different fresh stem cells in their natural environment along with numerous different type of supportive cells and growth factors
4- Good recovery not only Mesenchymal cells but also Very small embryonic-like stem cells (VSELs) which are the pluripotent stem cells with maximum regenerative potential.
5- Cells dont leave there biological environment.
6- Ficoll not used as it s biological effects on cells aging, damage to the genome (genotoxic ), changes in chromatin structure are still inconclusive. Possibility of mutations and late tumour formation
Bone Marrow Extraction–
Approx 120 to 240 ml is harvested from iliac crest at one time.
Aspirate one iliac crest on day 1, second iliac crest on day 15 and repeat the first iliac crest on day 30. Alternatively we can store the aspirate of Day 1 for future usages so that repeat aspirations are avoided
Bone marrow is processed in a closed system for volume reduction.
Approx 40-60 ml of the bone marrow is taken into the lab for expansion
Volume reduced sample is processed to have rich 2-5 ml of fraction of MNCs (haemopoetic stem cells and mesenchymal stem cells and endothelial progenitor cells) and adjoining supernatant plasma which is rich in platelets and growth factors.
The typical yield per 60 ml of bone marrow sample. The concentrate on an average contains 320-380 MIILION MNC, 15 to 18 million Cd 34+ve 10, 0000 to 15, 00000 MSC’s.
The sample is further processed to recover lost VSELS. This protocol is patent pending.
The Final volume is brought to 2to 20 ml depending on route of administration.
Adipose Tissue Extraction and Processing:
Using manual technique, 40 to 200 cc of fat is removed from abdomen. The fat is gently harvested to keep cells alive, using a medical procedure (not liposuction) blood and tumescent fluid layer is removed.. Wash fat with saline. Add human grade enzymes separating fat from stromal cells- Dissolve fat by massaging for 10 mins and allow to stand for 20 mins. The sample is then processed in Centrifuge 1000xg to get a pellet of stromal cells.
Stem cells from patient’s own fat /bone marrow/dental pulp/olfactory tissue umbilical cord tissue are grown in serum free media.
Passages only up to 3 times
The karyotyping of the alternate passage of cells showed a normal chromosomal pattern concluding their positive clinical usage.
Cells are then analyzed for viability and tested for stem cell specific characteristic surface markers and for bacterial, fungal and mycoplasmic burden in addition to the endotoxicity measurement
We prefer to inject these grown cells to Patient as fresh (directly from lab) without cryofreezing them. As extreme cold temperature effects efficacy and thawing reduces viability
Bio-activation – Stem Cells (either expanded or enriched) are bio activated with laser energy directed on them.
Results depend on Quantity and Quality of Stem Cells.
Quantity- the success rate depends on the amount of cells injected and the quality of cells. Ideal is-2-4 million mononuclear cells per kg body weight
Quality- is maintained by adhering to Good protocol
Stem cells are infused/injected weekly /fortnightly/monthly as per Different protocols. Total of 3-5 such infusions are suggested.
Route of Administration—
Optic atrophy and retinal diseases – Patients suffering from retinal and optic neuropathology may be benefited by a retro bulbar/ injection. The dose /quality and route is decided depending on the patient’s condition
1- Retrobulbar- for optic atrophy
2- Subscleral/ Intravitreal— for retinal diseases
3- Intrathecal – neurological disorders
4- Intravenous/ Intrarterial
6- Intralesional (direct)
2- Hyper Baric oxygen therapy
3- Intavenous and interstitial low level Laser
Vitamin B1 and Vitamin B12, Vit B3 nicotinic acid
Inj Adenosine Triphosphate